WO2002031206A2 - Methods and compositions using hybridization assays for detecting infectious agents - Google Patents

Abstract

A method for detecting the presence of target nucleic acid sequences in a human or animal subject infected by an infectious agent comprising obtaining a fluid or solubilized sample from the subject and detecting the presence of the target in the sample by nucleic acid hybridization. In addition, the present invention is directed to methods of detecting and measuring any oligonucleotides and polynucleotides of interest, including but not limited to mRNA for cytokines and hormones and genetic DNA sequences.

Description

METHODS AND COMPOSITIONS USING HYBRIDIZATION ASSAYS FOR DETECTING INFECTIOUS AGENTS

FIELD OF THE INVENTION

The present invention relates to the field of rapid detection and diagnosis of infectious agents utilizing nucleic acid hybridization to a biological sample obtained from a target infectious agent. In particular, the invention relates to methods of minimal sample preparation, and simple and reliable screening for hybridization of sample nucleic acids with labeled probes for target infectious agents, and other oligonucleotide and polynucleotide targets of interest.

BACKGROUND OF THE INVENTION

Viral strategies to evade a host's immune responses are many, complex, and ingenious (Whitton and Oldstone, 1996). Viruses can induce immune suppression by infecting macrophages, T and B cells to abrogate their function through one or several molecular mechanisms. For example, viruses can infect the thymus and thereby induce tolerance to the virus. Other viruses act by infecting antigen- presenting cells and destroying them. Still other viruses, such as influenza evade antibody responses by mutation (antigenic drift), or employ wholesale replacement of viral proteins by mixing genomic segments. Other strategies include blocking cell signaling and gene transcription factors, synthesis of soluble proteins that have binding sites for cytokines and chemokines, and downregulation of HLA Class I molecules (Michelson, 1999; otwal, 2000).

In response to viral or other foreign nucleic acids, infected host cells often release cellular products such as interferons. Interferons selectively block transcription and translation of viral RNA, stop viral replication without disturbing normal host cell functions and promote apoptosis in infected cells (Tanaka et al, 1998). Immune responses involving interferon have been shown in humans (Ghaziazdeh et al., 1997) and animals such as cows, pigs, chickens and cats (Chinsangaram et al., 1999).

Immune suppression continues to be a health and economic problem in the commercial production of poultry. Immune suppression by many agents has been reported but little is known about immune suppression of poultry in general. Furthermore, there is a lack of appropriate methods for evaluating the effect of immune suppression on general health, and its economic impact. Whereas much is known about the strategies viruses use to evade immune surveillance in other species, there is a paucity of information on chicken viruses. Again, this is partly because of the lack of appropriate methods to detect viral evasion of immune surveillance.

Methods for the detection of such virus-induced immune suppression are vital for the diagnosis and treatment of viral infections. The quest for rapid, facile, and accurate diagnosis of viral infections has, in recent years, focused on molecular nucleic acid assays. These methods are replacing formerly dominant immunologic tests because reliance of the latter on higher titers in convalescent serum interposes undesirable, and often unacceptable, delay in therapeutic intervention. Detection of nucleic acids has an important role in diagnosis of a variety of diseases in humans and in other animals. The conventional molecular techniques for detection of nucleic acids are usually expensive, time consuming and require laborious preparation of samples, including nucleic acid extraction. In addition, the use of harsh procedures results in destruction of cellular morphology, which is a great disadvantage in cases when cellular localization of specific sequences is of particular interest. Furthermore, the popular polymerase chain reaction (PCR) tests have potential for spurious results, as recently demonstrated (Dingemans et al, Laboratory Investigation 77, 213-20, (1997); Costa, Laboratory Investigation, 77, 211-2, (1997). In situ hybridization on tissue sections or cell preparations requires visual examination by light microscopy with attendant sample error. Moreover, quantitative measures cannot be made.

Chicken anemia virus (CAV) is an example of an infectious agent that is frequently tested for in large populations of chickens. Detection tests for CAV that are easy to administer and record are particularly desired. Although, in situ hybridization on blood smears for diagnosis of anaemia in chickens caused by CAV has been reported (Novak et al, Molecular and Cellular Probes 11, 135-41 (1997), Novaket al, Veterinarska stanica 27, 323-5 (1996); Sander et al, Avian Diseases, 41, 988-92 (1997), this testing procedure is rather cumbersome. Whereas this method is useful for testing a few samples collected from the field, it requires sensitive manipulations and visual examination by light microscopy.

CAV, formerly called chicken anemia agent (CAA), was first isolated by Yuasa et al. (Avian Diseases, 23, 366-385 (1979)) and its viral particles were found to contain circular single-stranded DNA (Gelderbloom et al, Archives of Virology, 109, 115-120 (1989); Todd et al, Journal of General Virology, 71, 819-823 (1990)). CAV causes severe aplastic anemia in young chickens (Yuasa et al, Avian Diseases, 366-385 (1979), Taniguchi et al, National Institute of Animal Health

Quarterly, Japan, 22, 61-69 (1982); Taniguchi et al, National Institute of Animal Health Quarterly, Japan, 23, 1-12 (1983)), poor growth, immune suppression, depletion of lymphoid organs, subcutaneous and intramuscular hemorrhages, and destruction of erythroblastoid cells in bone marrow (Bulow et al, Journal of Veterinary Medicine, B, 32, 679-

693 (1985), Yuasa et al, Avian Pathology, 16, 521-526 (1987), Jeurissen et al, Thymus, 14, 115-123 (1989)). Immune suppression caused by CAV infection is among the most important aspects of the disease (Otaki et al, Avian Pathology, 16, 291-306 (1987); Cloud et al, Immunopathology, 34, 3-4 (1992a); Cloud et al, Veterinary Immunology and Immunopathology, 34, 353-366 (1992b); Bounous et al, Avian Diseases, 39, 135-140 (1995)). Massive necrosis and apoptosis of lymphocytes causes immune suppression during the stages of disease when they are present. But, immune suppression often begins earlier, and almost always persists much longer, especially with CAV. In the field,

CAV causes more serious problems when associated with other viruses (Yuasa et al, Avian Diseases, 24, 202-209 (1980); Bulow, et al, Journal of Veterinary Medicine, B, 33, 717-726 (1986); Engstrom, Avian Pathology, 17, 23-32, (1988); Rosenberger, et al., Avian Diseases, 33, 753-759, (1989a); Rosenberger, et al, Avian Diseases, 33, 707-713,

(1989b)). Very little is known about how immune suppressive viruses of poultry cause immune suppression. It has been observed that interferon responses were depressed by CAV when either one-day-old (Adair et al., 1991) or three-week-old (McConnell et al., 1993) chickens were infected. Infection of chicks lacking maternal antibodies for CAV may be economically devasting because of high mortality. Chickens infected after the first week of age. have low mortality but significant economic losses are incurred from poor performance, presumed to be a result of immune suppression (McNulty et al, 1991). Current diagnostic tests for CAV are based on serological methods using neutralization tests (Bulow, et al, Journal of Veterinary Medicine, B, 32, 679-693 (1985), ELISA(Todd, et al, Journal of General Virology, 71, 819-823, (1990)) and immunofluorescence assays (McNulty, et al, Recent Advances in Virus Diagnosis, 15-26, (1984) (Bulow, et al, Journal of Veterinary Medicine, B, 32, 679-693 (1985) reported two additional tests: polymerase chain reaction (PCR) and a dot- blot assay, using digoxigenin-labeled c-CAV DNA as probe on CAN isolates propagated in the lymphoblastoid T-cell line, MDCC-MSB1 (Allan et al, Avian Diseases, 37, 177-182, (1993)) compared the immunocytochemical method for detection of CAV antibodies and in situ hybridization technique for localization of CAV on formalin-fixed and paraffin-embedded thymus tissue.

Other methods of choice in laboratory diagnosis of chicken anemia include isolation of CAV in cell cultures (Yusasa et al, National Institute of Animal Health Quarterly, Japan, 23, 78-81, (1983)) and the aforementioned immunofluorescence assays (McNulty et al, Recent Advances in Virus Diagnosis, 15-26, (1984)), ELISA's (Todd et al, Journal of General Virology, 71, 819-823, (1990)), and detection of virus by PCR and by in situ hybridization in formalin-fixed and paraffm- embedded thymus tissue (Allan et al, Avian Diseases, 37, 177-182,

(1993)). However, isolation of the virus is an extremely time-consuming procedure and not always the most appropriate because diagnosis is usually required in short period of time. In addition, immunofluorescence assays can be carried out only if specialized equipment is available as well experienced personnel, which is often not the case in diagnostic laboratories. Another disadvantage is that serological tests often have to be supplemented by antigen or nucleic acid detection in cases of inadequate immune response to acute infection by immunocompromised individuals.

When immunocytochemical methods are used for detection of virus in tissue, the main obstacle is cross-linking of proteins and subsequent masking of viral antigen as a result of routine formalin fixation of tissue following post mortem examination. The loss of antigenicity can be somewhat reversed by treatment with proteolytic enzymes but this is of limited use for viruses, such as CAV (Allan, et al, Avian Diseases, 37, 177-182, (1993)). In situ hybridization methods for detection of CAV in tissue sections, although not impaired following long fixation time, require conventional tissue preparation including paraffin embedment and tissue sectioning that delays diagnosis.

Although currently available molecular techniques for the detection of infectious agents are sensitive and specific, they often are technically complicated and expensive, especially when polymerase chain reaction must be used on each sample to be tested. Furthermore, currently available in situ hybridization detection assays are not particularly desirable because they are cumbersome in that technicians may have to handle numerous microscope slides and depend on visual interpretation for detection. Accordingly, presently available techniques and assays have potential for inaccuracy due to varying levels of technical skill and human interpretation of slides.

The need has emerged for tests for detecting the presence of infectious agents in humans or animals that can be easily automated and applied simultaneously to a large number of samples. There is also a need for a test that may enable the assessment of the immune status of an organism.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting the presence of an infectious agent in a human or animal. The invention further provides methods for detecting and measuring presence (and/or abundance) of mRNA for interferons as a measure of immune competence. The method comprises obtaining a sample from the human or animal and detecting the presence of a nucleic acid from the infectious agent by nucleic acid hybridization. The method involves diagnosis comprising competitive tests using nucleic acid. The assay may be conducted using double-stranded or single-stranded probes in microtiter plates, and the results can be measured with conventional plate readers. The novel design of the present invention enables a detection method that is fast, accurate, and may be easily quantified for estimating virus load.

The sample may comprise any biological tissue or body fluid, such as blood, saliva or tears or any solubilized specimen including extracts of any animal or plant tissue, water, sewage, chemicals, food and the like. The sample is prepared for hybridization with nucleic acid probes for the infectious agent of interest.

The infectious agent may comprise any pathogenic organism, including but not limited to, virus, bacteria, parasites, parvoviruses or other agent which infects a subject, for example human immunodeficiency viruses (HIV).

Additionally, the methods of the present invention can be used to detect and measure any ohgonucleotide or polynucleotide of interest.

In a preferred embodiment, a nucleic acid probe for infectious agent is immobilized and allowed to hybridize with the sample.

If sample is contaminated with the infectious agent, nucleic acid from the infectious agent will hybridize to the immobilized probe and compete with the labeled probe for hybridization sites on immobilized probe.

In another embodiment, the invention provides a method of detection for the presence of chicken anemia virus in a chicken comprising utilizing a preselected portion of a whole blood sample containing an intact blood cell, such as a lymphocyte obtained from the chicken and detecting the presence of a nucleic acid from chicken anemia virus in the intact blood cell by nucleic acid hybridization. In a another embodiment, CAV nucleic acid is detected in a lymphocyte containing peripheral blood smear or cytospin buffy coat preparation by in situ hybridization.

In yet another embodiment, the present invention provides a rapid, efficient method for determining the immune status of animals and humans. The invention provides a method of observing immune response kinetics in animals and humans and measuring the mRNA for alpha and gamma interferons (IFN) by competitive nucleic acid hybridization.

The methods described herein are easy to use and inexpensive. The results obtained are both reliable and reproducible. The methods are cost-effective because equipment already present in most laboratories may be used.

The nucleic acid hybridization assays of the present invention may be conducted in microtiter plates of the present invention allowing for rapid testing of numerous samples and minimizing opportunities for error. The present study describes competitive hybridization assay techniques for detection of oligonucleotides and polynucleotides in samples. The methods described herein utilize simple and cost effective sample collection and preparation. In addition the methods are applicable for wide use in the field for rapid diagnosis of any infectious agent present in a variety of samples including body fluids, chemicals and other solubilized specimens.

Accordingly, it is an object of the present invention to provide compositions, methods and devices for detecting the presence of infectious agents such as bacteria, viruses and parasites. It is another object of the present invention to provide a rapid molecular test for determining the immune status of animals and humans.

Another object of the present invention is to provide a routine qualitative assay for the detection of infectious agents, such as CAV and HIV, that is easy, fast, accurate and provides reproducible results.

It is yet another object of the present invention to provide a quantitative detection assay for the detection of infectious agents for estimating infectious agent load, and for enabling the correlation of nucleic acids detection in biological samples, and the incidence of disease in normal, healthy individuals, or immunocompromised subjects.

Still another object of the present invention is to provide a detection assay for infectious agents by detecting oligonucleotides and polynucleotides. Another object of the present invention is to provide compositions, methods and devices for screening markers of infectious agents by detecting oligonucleotides and polynucleotides.

A further object of the present invention is to provide a kit containing an optimized assay configuration for automated pointof-use analysis for detecting oligonucleotides and polynucleotides of infectious agents in biological samples.

An additional object of the invention is to provide a method of detection of infectious agents for monitoring the effectiveness of therapeutic treatments.

Yet another object of the invention is to provide methods that are particularly suited for rapid throughput and screening of samples, utilizing equipment already used in a laboratory for screening, without the need for extensive training or expensive laboratory equipment. Another object of the present invention is to provide methods for rapid detection of infectious agents that overcome the problems of the prior art, such as those caused by hemoglobin interfering with PCR assays.

It is an object of the present invention to provide compositions, methods and devices for detecting the presence of infectious agents such as bacteria, viruses and parasites without the use of radioactivity.

These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of a competitive nucleic acid hybridization assay in microtiter plates. Diagram A demonstrates immobilized capture probe limiting for labelled detection probe (L). Diagram B demonstrates that the binding of unlabelled standard or test sample DNA or RNA blocks the binding sites for L, resulting in proportional decrease in signal. Figure 2 is a log linear graph depicting color development detected in a competitive nucleic acid hybridization assay: absorbence against increasing amounts of competing sample.

Figure 3 is a graph showing titration of immobilized capture probe in microtiter plates. As further described in the examples, to determine the amount of immobilized probe that would give signal in a desirable range, after hybridization with labeled probe, 0, 50, 75, 100, 125, and 150 ng of the probe were immobilized onto nitrocellulose discs. The discs were incubated in prehybridization cocktail in microtiter plate wells, followed by incubation in hybridization cocktail containing 1 μg ml^"1 of denatured, biotinylated probe. Discs were washed, an avidin-biotin-alkaline phosphatase complex applied, discs washed, p- nitrophenyl substrate added, and product measured at 405 nm. Points are means of triplicates. The curve is a smoothing spline fit at λ=l 0,000 (r²=0.98).

Figure 4 is a graph providing competition curves for chicken anaemia virus DNA (standard curves) and infected MSB1 DNA (samples). The standard curves are on the left side and sample curves on the right side. Numbers match curves done concurrently with the same probe, and they are the same as in Table 1.

Figure 5 is a graph providing pooled competition curves for chicken anaemia virus DNA standards (left side) and infected MSB 1 DNA (right side) for data in Fig. 4 and Table 1 excluding data from test 3. Broken lines are 95% CI. Figure 6 provides two graphs showing binding of labeled

DNA probes to 100 ng corresponding, immobilized DNA capture probes for chicken interferon alpha and gamma mRNA. Broken lines are 95% confidence intervals. The r for both curves was 0.98.

Figure 7 is a graph showing the kinetics of induction of mRNA for interferon alpha in chickens challenged with inactivated

Newcastle disease virus (Table 6). Decrease in signal reflects increase in IFN mRNA. Plot is a smoothing spline fit at λ= 0.01, r ²=1.35.

Figure 8 is a graph showing the kinetics of induction of mRNA for interferon gamma in chickens challenged with inactivated Newcastle disease virus (Table 6). Decrease in signal reflects increase in

IFN mRNA. Plot is a smoothing spline fit atλ = 0.01, r²=0.98. DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of specific embodiments included herein. Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention. The entire text of the references mentioned herein are hereby incorporated in their entireties by reference. As used in the claims, "a" may mean one or more than one, depending upon the context within which it is used. The term "in situ hybridization" (ISH), as used herein, includes any of the methods known in the art for detecting the presence of specific nucleic acid sequences in cells including, but not limited to techniques described in (Mitchell et al, Medical Laboratory Sciences, 49, 107-118, (1992)) and (Martinez et al,

Journal of Histiochemistry and Cytochemistry, 43, No.0, pp 0-00, (1995)).

As used herein, the term "nucleic acid" is intended to encompass all genetic matter found in an organism, including but not limited to oligonucleotides and polynucleotides.

The present invention provides a method for detecting the presence of an infectious agent in a sample from a subject. Nucleic acid hybridization is employed to detect the presence of a nucleic acid from the infectious agent in the sample. In one embodiment, the method for detection of the infectious agent comprises contacting the sample with at least one detectable nucleic acid probe that is selective for the agent under conditions favorable for promoting hybridization of the probe to a sample. If the sample contains the infectious agent, the probe will bind to the sample. The presence of the hybridization between the probe for the infectious agent and the sample is detected, thereby detecting the presence of the predetermined infectious agent in the sample.

The method can specifically comprise the steps of contacting a preselected portion of a blood sample with at least one detectable nucleic acid probe selective for an infectious agent (such as chicken anemia virus) under conditions which are favorable for promoting hybridization of the probe to the infectious agent nucleic acid, and detecting the presence of the hybridization between the probe and the infectious agent nucleic acid, thereby detecting the presence of the infectious agent in the sample. An additional use of the methods described herein provides a means for studying viral evasion of immune surveillance, and as a test for assessing immune status in humans and animals, especially commercial chickens. Transcription of human IFN-γ has been correlated with secreted rNF-γ and with immune responsiveness. Since such a challenge antigen will be processed and presented rapidly with abundant transcription of mRNA in several hours, this increase can be used to monitor immune status.

The test is based on abundance of mRNA for alpha and gamma interferons (IFN) induced by immune challenge such as CMV or inactivated Newcastle disease virus (iNDV). This induction is the earliest hallmark of an immune response. Total RNA is extracted from whole blood, and IFN mRNA is measured by competitive nucleic acid hybridization in microtitre plates that employ manipulations similar to ELISA. The test does not require isolation of the mRNA fraction, and it can be done using total RNA.

The quantification of IFN mRNA can serve as a diagnostic tool for virally induced immune suppression, since induction of IFN mRNA can be observed on the basis of absorption. This can be done with the described test providing the sample in question has a slope that is not different from a standard curve. Plasmid probe cannot be used as standard because it contains plasmid DNA. Standards produced by RT-PCR can be used since they do not contain plasmid DNA.

Samples The methods of the present invention may be used to detect infectious agents in any solubilized sample. Samples can be any biological tissue or body fluids, including, but not limited to virus stock samples, serum, cells, plasma, semen, urine, saliva, sputum, blood, tears, mucus and cerebrospinal fluid. In addition, samples may include non- biological samples such as water (from lakes, rivers, swimming pools etc.), and other substances such as beverages. As used herein, the phrase "preselected portion of a whole blood sample" is meant to include, but not be limited to any portion of a blood sample obtained from a human or animal subject which contains at least one detectable intact blood cell. The blood cell can be any blood cell including, but not limited to red blood cells and their precursors, white blood cells such as neutrophils and lymphocytes, thrombocytes and platelets, macrophages and the like. In one embodiment, the preselected portion of the blood sample is a peripheral blood smear. In yet another embodiment, the preselected portion is a cytospin buffy coat preparation or an intact lymphocyte.

Nucleic Acid Probes Derived from Target Infectious Agent

One embodiment of the present invention utilizes synthetic oligonucleotide probes which are selective for infectious agents such as chicken anemia virus. Examples of such probes include, but are not limited to the nucleotide sequences set forth in the Sequence Listing as SEQ ID NO:l to SEQ ID NO: 12. It is specifically contemplated that the probes of SEQ ID NO:l to SEQ ID NO: 12 can be used alone or in combination with each other or another suitable probe in the above methods.

In general, primers and probes for PCR, LCR and in situ hybridization are usually about 20 base pairs in length and the preferable range is from 15-25 base pairs. However longer probes can be utilized. Methods for synthesizing probes and primers from known conserved sequences of the predetermined infectious agent include production by recombinant technology and other methods known in the art (Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), (Piper and Unger, A Primer for Pathologists, ASCP Press, Chicago, (1989)). Better amplification is obtained when both primers are the same length and with roughly the same nucleotide composition. Denaturation of strands usually takes place at 94° C and extension from the primers is usually at 72° C. The annealing temperature varies according to the sequence under investigation. Examples of reaction times are: 20 mins denaturing; 35 cycles of 2 min, 1 min, 1 min for annealing, extension and denaturation; and finally a 5 min extension step. Conditions favorable for hybridization of probes to a target nucleic acid are known in the art, (see for example Allan et al, Avian Diseases, 37, 177-182, (1993), Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1982), Mitchell, et al, Medical Laboratory Sciences, 49, 107-118, (1992), Piper and Unger, A Primer for

Pathologists, ASCP Press, Chicago, (1989), and are set forth in the examples.

The probes provided herein may be suitably labeled with, for example, a radiolabel, digoxygenin-label, enzyme label, fluorescent label, biotin-avidin label and the like for subsequent visualization.

Specific examples are set forth in Mitchell et al, Medical Laboratory

Sciences, 49, 107-118, (1992).

Infectious Agents The "infectious agent" can be any infectious agent which is, or can be present within a sample including, but not limited to bacteria, viruses and parasites. Examples of such infectious agents include HIV virus, rotavirus, chicken anemia virus, equine infectious anemia virus, various bacterial pathogens such as Escherichia coli and Salmonella and blood borne pathogens such as Anaplasma sp., Babesia sp. and Rickettsia sp. Other examples of infectious agents are known in the art and can be found for example, in Piper and Unger.

Hybridization Conditions which are favorable for promoting hybridization of the particular probe to the nucleic acid of the infectious agent can vary depending upon the infectious agent or sample being tested or the type of probe utilized. However, such conditions are generally known in the art and will be apparent to the skilled artisan (see for example Allan et al, Avian Diseases, 37, 177-182, (1993), Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1982), Mitchell, et al, Medical Laboratory Sciences, 49, 107-118, (1992), Piper and Unger, A Primer for Pathologists, ASCP Press, Chicago, (1989), Martinez et al, Journal of Histiochemistry and Cytochemistry, 43, No. 8, pp 739-747, (1995)). Thus, one can merely adapt the procedures set forth in the

Examples to suit the preselected infectious agent.

The probe can be immobilized on a substrate and the sample hybridized thereto. The substrate can be anything currently used in the art, such as nitrocellulose filters, microtiter plates, etc.

Alternatively, the sample can be in solution and the probe added thereto under conditions favorable to hybridization.

The type of hybridization is chosen based on the type of sample and probe. For example, competitive hybridization can be used for rapid, sensitive detection of the amount of infectious agent. In situ hybridization can be used for detection of the presence and localization of infectious agents in tissues and cells. ELISA is used for detecting an antibody response to an infectious agent, however, ELISA results do not reveal the cause of an antibody response, i.e., vaccine, infection etc. Other examples of hybridization and when each is used are well known in the art and some are provided in Example 5.

Detection

The present invention specifically contemplates all forms of specific nucleic acid sequence detection which can be adapted to be performed on samples containing nucleic acid. A preferred embodiment utilizes nucleic acid probes and competitive hybridization for the detection of nucleic acid from a predetermined infectious agent from a solubilized sample. Biotin labels are used and colorimetric detection is employed. The methods of the present invention can be used for the detection of single or double stranded nucleic acid. The assay is particularly desirable in the embodiment wherein a double stranded probe is used to detect the double stranded nucleic acid of an infectious agent.

Other methods of nucleic acid detection known in the art such as polymerase chain reaction (PCR) with or without restriction fragment length polymorphism (RFLP) analysis, ligase chain reaction, and PCR reaction of specific alleles (PASA) can be utilized to enhance the subject assay and are described for example in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1982). Quantification

The target infectious agent load can be measured from a standard curve. For example, whenever an amount of nucleic acid is known to represent a specific viral dose, i.e. number of virions, infectious dose units, etc. the viral load (in those units) is revealed by the corresponding nucleic acid measured by the present invention.

Immune Status

The competitive nucleic acid hybridization method described herein may be used to evaluate immune status in a human or animal. The competitive nucleic acid hybridization tests for interferon (IFN) alpha and gamma mRNA can be done in microtitre plates using total RNA contracted from circulating blood preserved in the cationic detergent Catrimox-14. The impairment of IFN mRNA transcription was found to be paralleled by immune responses to iNDV. Thus, the derived test for abundance of IFN mRNA can be used to investigate interference with transcription by viruses that cause immune suppression. Furthermore, the test in combination with iNDV challenge, can be used to assess immune status of commercial poultry.

Embodiments

One embodiment of the competitive nucleic acid hybridization assay (Example 5) involves hybridizing sample DNA to "capture", or probe, DNA that has been immobilized onto nitrocellulose, followed by hybridization of biotinylated probe to the capture probe DNA in a microtiter plate. In this design, the labeled (biotinylated) probe is in excess and capture DNA probe is limiting. Alternative embodiments include assays wherein sample DNA is hybridized to the labeled probe in solution to occupy sequences in the labeled probe. (Example 6) Then the labeled probe, that now has fewer sequences available for binding to capture DNA, can be applied to the immobilized capture DNA under conditions wherein the capture DNA is in excess, and the labeled probe is limiting. If the immobilized capture DNA and the soluble, labeled DNA are equimolar, the competition may be performed against either component. The microtiter assay may be used with fluid samples such as blood, soluble extracts, solubilized extracts of any tissue, animal or plant, water, sewage, manufacturing process components, foods containing nucleic acid sequences of interest, including but not limited to nucleic acid of virus, bacteria or other organism of interest.

The present invention further provides a kit for detecting particular infectious agents in biological samples by competitive hybridization of nucleic acids comprising enzymes, buffering agents, cations and oligonucleotides well known in the art for preparing probes and for carrying out hybridization and detection reactions. In one embodiment of the kit, the kit comprises substrates on which to immobilize the probe or sample, such as microtiter plates and nitrocellulose. Also included are enzymes, buffering agents and other components necessary for immobilizing the probe or sample. The kit also comprises suitable reagents for detecting the infectious agent. The reagents included in the kit will depend on which type of hybridization the kit is designed to facilitate. The components can each be in separate containers or all or any combination of these components can be combined in a single container. The complementary DNA primers for PCR or the nucleic acid probes can be the oligonucleotides of SEQ ID

NOS:l-12.

This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention.

Example 1

In situ hybridization for detection of CAV in blood smears

Method

Preparation of Samples Blood smears from known CAV positive and known CAV negative chickens were obtained from SPAFAS, Inc. (Storrs, CT). Blood smears were fixed in 4% paraformaldehyde for 30 minutes, washed in TRIS-saline (0.1 M TRIS-Cl pH 7.5, 0.1 M NaCl), and dehydrated in graded 30, 60, 80, 95, 100% ethanol series. Dry slides were stored in sealed container at 4°C prior to hybridization. The ability to interact with hybridization probe was not adversely affected by keeping slides stored under these conditions.

Preparation and labeling of nucleic acid probe from CAV

The oligonucleotide sequences were derived from the CAV DNA sequence described by Noteborn et al, Avian Pathology2l,

107-118, (1992), and synthesized at the Molecular Genetics Facility at the University of Georgia.

First Probe: A cocktail of two synthetic oligonucleotides, 5' TCG CAC TAT CGA ATT CCG AGT G 3' set forth in the Sequence listing as SEQ ID NO:l and 5' GGC TGA AGG ATC CCT CAT TC 3', set forth in the Sequence Listing as SEQ ID NO.J was used for in situ hybridization. The oligonucleotides were digoxigenin and biotin labeled by 3' end-labeling reaction described by Maniatis et al, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1982). Hybridization cocktail containing

45% formamide was purchased from Amresco (Solon, OH). 0.5 μg/ml labeled probe was added to the hybridization cocktail.

Second Probe: The same DNA fragment of CAV was also amplified by PCR using oligonucleotide primers 5' TCG CAC TAT CGA ATT CCG AGT G 3' (SEQ ID NO: 1) and 5' GGC TGA AGG ATC

CCT CAT TC 3' (SEQ ID NO:2). The template for amplification of probe by PCR was DNA extracted from MDCC-MSB1 cells using proteinase (1 mg/ml proteinase K, 1 mM EDTA, 10 mM TRIS, pH 8, 1% SDS) for 2 hours at 37°C, followed by extraction with phenol:chloroform:isoamyl alcohol (25:24:1), chloroform:isoamyl alcohol (24:1), and precipitation with ethanol at -20°C. Polymerase chain reaction was carried out in 100 μl volumes, which included 0.5μg of each primer, 10 μl of extracted DNA, deoxyribonucleotides in final concentration of 100 μM each (Pharmacia, Piscataway, NJ), Taq polymerase (Promega, Madison, WI), and 2 mM MgC^ A Perkin-Elmer thermal cycler was programmed to carry out a 2 minute denaturation step at 94°C, followed by 25 cycles composed of 2 minutes at 94°C, 1 minute at 47°C, and 3 minutes at 72°C. A final extension period of 7 minutes at 72°C was followed by storage at 4°C. Amplified DNA was extracted from the PCR reaction mixture following the same procedure described for extraction of DNA from CAV infected cells omitting incubation with proteinase K. The amplified fragments of CAV DNA were labeled with digoxigenin-lldUTP (Boehringer Mannheim, Indianapolis, IN) or biotin- 11-dUTP (Boehringer Mannheim) by the nick translation method (von Bulow, et al, Zentralblatt fur Veterinar Medizin B, 33:93-116, (1986)). Unincorporated nucleotides were removed by gel filtration using Quick

Spin Columns (Boehringer Mannheim). The integrity of PCR product and success of the labeling procedure were confirmed by dot-blot hybridization (von Bulow, et al., Zentralblatt fur Veterinar Medizin B, 33:93-116, (1986)) of known CAV DNA template using uninfected cell DNA as negative control.

Pretreatment for in situ hybridization

Blood smears were hydrated in graded 100, 95, 80, 60, 30% ethanol series, soaked briefly in TRIS-saline, and placed for 10 minutes in 0.02 N HC1. After two washes in TRIS-saline, 3 minutes each, slides were incubated 1.5 minutes in PBS containing 0.0l°A> Triton X-100. Following two washes in TRIS-saline, lasting 3 minutes each, the slides were incubated for 5 to 10 minutes (digestion was controlled visually under a microscope) in pronase (0.5 mg/ml) dissolved in 0.05 M Tris-HCl buffer, pH 7.6, containing 5 mM EDTA. After washing twice for 3 minutes in TRIS-saline containing 2 mg/ml glycine, smears were post- fixed in 4% solution of paraformaldehyde in TRIS-saline, washed twice for 3 minutes in TRIS-saline with 2 mg/ml glycine, and dehydrated through a graded ethanol series (30, 60, 80, 95, and 100%- twice each for 5 minutes) and finally air dried.

Hybridization

The hybridization cocktail previously prepared was spotted over the blood smears and covered with autoclavable coverslips. Smears and the probe were denatured by heating the slides at 110°C for 10 minutes. The slides were then incubated for two hours at 37°C. When hybridization was complete, slides were dipped in 2X SSC and first washed twice in 2X SSC 0.1% SDS, twice in 0.2X SSC 0.1% SDS, twice in 0.1X SSC 0.1% SDS, for 3 minutes each step. Next they were washed once for 1 minute in 2X SSC 0.1% SDS, and then incubated for 5 minutes in 3% BSA in TRIS-Cl, pH 7.5, 0.1 M NaCl, 5 mM MgCl2, 0.25% Brij

(TRIS-saline Brij, pH 7.5). Following the wash steps the slides were air dried.

Colorimetric detection Blocked blood smears, hybridized with biotin labeled probe, were incubated for 5 minutes at room temperature in an avidin- alkaline phosphatase complex. The avidin-alkaline phosphatase complex had been freshly prepared by combining 40 μm Avidin DN (Vector Laboratories, Burlingame, CA), 5 μl of biotinylated alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN) and 11 ml 1% BSA in TRIS- saline Brij, pH 7.5. Blood smears, hybridized with digoxigenin-labeled probe, were incubated for 20 minutes at 37 C with anti-digoxigenin- alkaline phosphatase Fab fragments (Boehringer Mannheim) diluted 1:600 in TRIS-saline Brij, pH 7.5, containing 1% BSA. Smears, hybridized with biotin- and digoxigenin-labeled probes, were after this step washed three times for 3 minutes in TRIS- saline Brij, pH 7.5, and once in TRIS-saline Brij pH, 9.5 (0.1 M TRIS-Cl, pH 9.5, 0.1 M NaCl, 50 mM MgCfe). Color was developed by incubating slides for 2 hours at 3 / C according to McGadey Histochimie 23, 180-84 (1970). The McGadey reagent had been freshly prepared by adding 67 μl of 50 mg/ml 50% N,N- dimethyl formamide stock solution of nitro blue tetrazolium and 33 μl of 50 mg/ml 50% N,N-dimethyl formamide stock solution of 5-bromo-4-chloro-3-indoyl phosphate p- toluidine salt to 10 ml TRIS-saline, pH 9.5. The slides were washed three times for 1 minute in TRIS- saline, pH 7.5, counterstained with nuclear fast red, air dried, and mounted in 70% Permount (Fisher Scientific, Norcross, GA) diluted with xylene. Results

Following processing by in situ hybridization, blood smears obtained from chickens infected with CAV contained positive lymphocytes with clearly noticeable signal produced by alkaline phosphatase with McGadey reagent in the form of dark-blue formazan crystals. Due to relatively weak signal, as a result of using cocktail of synthetic oligonucleotides as a probe, signal developed slowly. The optimal ratio between signal and background was obtained after two hours of incubation at 37 C. Hybridization was considered positive when one or more cells had unambiguous nuclear or cytoplasmic staining.

Nonspecific staining, when it occurred, consisted of dark dots in a random pattern dispersed in spaces between blood cells.

Blood smears from uninfected chickens occasionally showed some nonspecific staining, but on higher-power examination, this staining was easily distinguished by its extracellular location. To exclude any staining possibly due to endogenous alkaline phosphatase activity, the avidin-enzyme complex was omitted from colorimetric detection procedure. Blood smears stained following this procedure showed no color development. Thus, in situ hybridization for detection of CAV in blood smears, in addition to providing rapid results, requires very simple sampfe collection and is convenient for quick screening of large numbers of birds.

Example 2

In situ hybridization for detection ofretrovirus HIV in cytospin preparations

Method Preparation of Samples

Cytospin preparations of peripheral blood monocytes from chimpanzees that were infected and those that were not infected with

HIV- 1 were prepared on glass microscope slides. Cytospin preparations also were prepared with cultured cells infected with one copy of HIV-I or HTLV-1. The cytospin preparations were fixed in paraformaldehyde, treated with pronase and postfixed with paraformaldehyde as described in example for CAV.

Preparation and labeling of nucleic acid probe from HIV-1 Selected oligonucleotides specific for the retrovirus HIN were synthesized with biotin bound at the 5' end. The sequences were: oligonucleotide 1, 5' ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AGC CCT AC 3' (SEQ ID ΝO:3); oligonucleotide 2, 5' CAA TGA GAC ACC AGG GAT TAG ATA TCA GTA CAA 3' (SEQ ID NO:4); oligonucleotide 3, 5' ATG GGT GCG AGA GCG TCA GTA TTA

AGC G 3' (SEQ ID NO:5); oligonucleotide 4, 5' AAT CCT GGC CTG TTA GAA ACA TCA GAA G 3' (SEQ ID NO:6): oligonucleotide 5, 5' CGC TTA ATA CTG ACG CTC TCG CAC CCA T 3' (SEQ ID NOJ); oligonucleotide 6, 5' GGG AGC TAG AAC GAT TCG C 3' (SEQ ID NO:8). A cocktail of all six oligonucleotides was made in water and contained 0.167 μg/μl of each oligonucleotide.

Hybridization and Colorimetric Detection

Hybridization and color detection were as described in example for CAN.

Results

Positive staining was observed by light microscopy on cells from infected chimpanzees and cells infected in culture with HIV- 1, but not from uninfected chimpanzees or cells infected in culture with

HTLV- 1 .

Example 3

Competitive spectrophotometric detection of target nucleic acids in microtiter plates having immobilized unlabeled probe

Method

Preparation of samples

Anticoagulated blood samples (500 μL) were mixed with 1 ml of 37.5 mM ΝaCl and centrifuged at 800g for 5 minutes at 4°C to collect cells at the bottom of the tube. The supernatant was discarded and the pellet was washed several times with the salt solution to eliminate the majority of hemoglobin. Cells suspended in 37.5 mM NaCI were then frozen and thawed. 100 ul of 0.5 M NaOH and 5M NaCI were added to 400 μl of the sample to release and denature the DNA. The samples were mixed well and incubated for one hour at 37°C.

Preparation of unlabeled nucleic acid probes from a target infectious agent

Unlabeled nucleic acids from a target infectious agent were immobilized on microtiter plates. An unlabeled probe for the nucleic acid of interest must first be denatured prior to immobilization. When the unlabeled probe is DNA, it is may be denatured by two different methods. In the first method denaturing is done by the boiling of DNA suspension (1 μg/50μl) in TE buffer (lOmM Tris5 ImM EDTA) for 10 minutes in a water bath. In a second method the DNA suspension is first combined with 40 μl (20 μg) of DNA probe, 75 μl 2M NaOH and 385 μl dH2θ, next it is heated at 65°C for one hour, cooled to room temperature and finally 500 μl 2M ammonium acetate, pH 7.0 is added.

Denaturing of an unlabeled RNA probe is most commonly achieved by combining 10 μl suspension of RNA (2.4 μg dissolved in dH2O) with 20 μl 100% formamide, 7 μl 37% formaldehyde, and 2 μl

20X SSC. The mixture is then incubated at 68°C for 15 minutes, followed by cooling on ice. Two volumes (78 μl) of 20X SSC are added to complete the denaturing process. To immobilize denatured nucleic acids in the wells, 1 μg of denatured nucleic acid (50 μl) is pipetted into the well and 50 μl of immobilization buffer (1.5M NaCI, 0.3M TrisHCI, pH 8.0; 0.3M MgCl2) is added. Plates are incubated overnight at 37°C. The denatured nucleic acid mixture is then removed from the wells, and the wells are allowed to dry for 30 minutes at 37°C. The wells are irradiated at 254 nm and washed three times with washing buffer (1M NaCl; 0.1M Tri-HCl, pH 9.3; 2mM MgCl2; 0.1% Tween 20). Other methods of immobilization are well known to those of ordinary skill in the art.

Wells containing immobilized, denatured nucleic acids can be used immediately or stored at 4°C in a sealed bag. Prehybridization

To each well of microtiter plate was added 100 μl of prehybridization solution containing 6X SSC (0.9 M NaCl, 0.09 M Na citrate), 5X Denhardt's solution, 0.01 M EDTA and 0.5% SDS. The plates were then incubated for one hour at 56°C.

Hybridization

Hybridization of sample nucleic acid with immobilized unlabeled probe A mixture consisting of 1J3 ml deionized 100% formamide, 0.96 ml 20X SSC, 38 μl 100X Denhardt's solution, 154 μl 0.5

M Na phosphate, pH 6.5, 77 μl of 10 mg/ml freshly denatured sheared herring sperm DNA, 385 μl 50% dextran sulfate, and 400 μl of sample,

(as described above) was prepared. 100 μl of the mixture was next pipetted into each well and the plates were then incubated for two hours at

56°C. After hybridization, the hybridization cocktail was discarded and the plates were washed three times for 3-5 minutes at room temperature in

2X SSC, 0.25% Brij, and 0.1% SDS followed by two washes in 0JX

SSC, 0.25% Brij, 0.1% SDS at 56°C for 10-20 minutes. The plates were then rinsed in prehybridization solution.

Hybridization of labeled probe with immobilized unlabeled probe

100 μl of hybridization cocktail containing 45% formamide, 5X SSC, IX Denhardt's solution, 20 mM sodium phosphate, pH 6.S, 0.2 mg/ml freshly denatured sheared herring sperm DNA, 5% dextran sulfate, and 0.5 μg/ml biotinylated probe was then added to the plates. After incubation at 56°C for two hours, the hybridization cocktail was removed from the plates and the plates were rinsed briefly with 2X SSC. The plates were washed two times for 5 minutes each in 2X SSC, 0.25% Brij, and 0.1% SDS and two times for 10-20 minutes each at 56°C in 0.2X SSC, 0.25% Brij, and 0.1% SDS.

Next 2% BSA in TS Brij, pH 7.5 was added to the plates. After incubation at room temperature for 10 minutes the BSA solution was discarded and 100 μl per well of avidin-biotin-alkaline phosphatase complex (40 μL of 1.0 mg/ml avidin and 5 μl of 2000 U/ml of biotin-alkaline phosphatase in 2% BSA in TS Brij, pH 7.5) was added The plates were then incubated for 10 minutes at room temperature.

Finally the plates were washed three times in TS Brij pH 7.5 (0.1 M Tris-Cl pH 7.5, 0.1 M NaCl, 5 mM MgCl2, 0.01% Brij), and once in TS Brij, pH 9.5 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM

MgCl₂).

Colorimetric Detection

Preparation of the substrate solution consisted of mixing p-nitrophenyl phosphate (0.75 mg/ml) in diethanolamine- HC1 buffer, pH

9.8 (0. lg MgCl2 X 6H2,0 and 96 ml diethanolamine in 11 dH2θ). 100 μl of substrate solution was added per well and incubated at room temperature for one hour. The absorbance was measured at 405 nm. Amounts of immobilized, unlabeled probe and labeled soluble probe were adjusted to obtain optimal signal (absorbance, fluorescence, chemiluminescence and the like, depending on labeling system used) which for absorbance systems is between 0J - 2.0 absorbance units. Positive control (nucleic acid extract of cells infected with virus) and test sample, if containing viral nucleic acid, cause significant decrease in absorbance by competition with soluble, labeled probe, whereas negative control (nucleic acid extract from uninfected cells ) does not.

Example 4

Development of Competitive Nucleic Acid Hybridization Assays in Microtiter Plates

Methods

The first step was to determine the amount of capture probe DNA immobilized on nitrocellulose that would, after hybridization, enable a degree of color development that can most accurately be read by a standard microtiter plate reader (< 1 absorbance unit). To begin with, 25, 50, 75, 100, and 125 ng of denatured CAV DNA was immobilized on nitrocellulose discs. The discs were placed in microtiter plate wells and prehybridized for two hours at 56°C in prehybridization cocktail containing 45% deionized formamide, 5X SSC, 25 mM sodium phosphate, Denhardt's solution, and 0.25 mg/ml sheared herring sperm DNA. Prehybridization cocktail was then replaced with hybridization cocktail containing 45% deionized formamide, 5X SCC, 25 mM sodium phosphate, Denhardt's solution, 25 mg/ml sheared herring sperm DNA, 10% dextran sulfate, and 0J μg/ml of biotinylated denatured CAV DNA probe. Hybridization proceeded overnight at 56°C.

After removing the hybridization cocktail, wells containing nitrocellulose discs were rinsed 2X SCC, washed twice in 2X SSC, 0.25%) Brij, and 0.1% SDS for 10 minutes at room temperature, twice in 0.2X SSC, 0.25% Brij, and 0.1% SDS for 10 minutes at room temperature, followed by two washes in the same solution for 15 minutes at 60°C. The discs were then incubated in blocking solution containing 1% BSA in 0.1M Tris-HCl, pH 7.5, 0.1M NaCl, 5 mM MgCl2 0.25% Brij

(Tris-saline Brij, pH 7.5) for 30 minutes at 56°C. Avidin-biotin conjugated with alkaline phosphatase complex was applied to wells and allowed to bind to biotinylated probe for 30 minutes at room temperature.

Wells were washed three times for 10 minutes at room temperature in Tris-saline M Tris-HCl, pH 9.5, (01 M NaCl, 50 mM MgCfe 0.25%

Brij). Then, 100 μl substrate solution, containing p-nitrophenyl phosphate (0J5 mg/ml) in diethanolamine-HCl buffer, pH 9.S (0.1 g MgCl2 x 6H2O, 96 ml diethanolamine, 11 ml dH2θ), was added to each well, and the plate was incubated in the dark for 30 minutes at room temperature.

Absorbance was measured at 405 nm in a conventional plate reader. From a plot of the data, 100 ng DNA immobilized on nitrocellulose disc, when hybridized with 200 ng/ml of biotinylated probe

(probe in excess) produced absorbance of between 0.9 and 1.0 O.D.

Second step: for the competitive nucleic acid detection assay, 100 ng of unlabeled CAV DNA (capture DNA) was immobilized per nitrocellulose disc. Discs were placed in wells of microtiter plates and prehybridized as described above. To determine sensitivity and range of competing DNA (sample DNA), we hybridized the immobilized capture DNA with 0, 5, 10, 20, and 30 ng of CAV DNA. Hybridization with 100 μl/ml of biotinylated CAV probe followed. Both hybridization steps, subsequent washing, and development of color were performed as described above. Results

A typical plot is shown in Figure 1. The slope was linear with a high coefficient of regression. About 2.5 ng DNA can be detected by the assay in its present configuration. Fluorescence or chemiluminescence methods may be used to increase sensitivity by one log, or more, if necessary. The nanogram range probably is satisfactory for productive viral infections.

The nitrocellulose discs were used to show feasibility. Other solid phase matrices can be used, such as in the well (necessitating a transfer to another plate if matrix material is opaque or interferes with signal detection in some other way), or by placing immobilized phase on a plastic tab that extends down from a lid into the well, the lid may then be discarded and the rest of the test run in the original microtiter plate.

Example 5

Competitive detection of target infectious agents in microtiter plates having immobilized unlabeled probe

Method Preparation of samples

One-day-old, pathogen-free chickens, hatched from eggs obtained from SPAFAS, Inc. (Storrs, CT, USA), were divided into two groups, 50 birds each. One group was injected intracoelomically with 0.1 ml of CL-1 strain of CAV having a titer of 10⁶ TCID₅₀ (Yuasa et al., National Institute of Animal Health Quarterly, Japan 23, 75-7 (1983)), while another group from the same hatch was not injected. The two groups were housed separately in Horsfal Bauer units supplied with filtered air under positive pressure. Water and food were provided ad libitum. On the initial day of experiment (day 0), 3 birds were bled by cardiac puncture before injection of CAV to obtain samples for haematocrit, virus isolation, ELISA, dot-blot, and competitive nucleic acid hybridization microtiter plate test (MPT). Samples from 3 birds in each group were collected on days 2, 3, 4, 5, 7, 11, 13, 17, 19, 21, and 28. Heart blood was drawn into 6 mg ml^"1 EDTA at a ratio of 1:0.15 for all tests except haematocrit. Preparation and labeling of nucleic acid probes

A DNA fragment of CAV was amplified by PCR using oligonucleotide primers 5'-TCG CAC TAT CGA ATT CCG AGT G-3* (SEQ ID NO:l) and 5'-GGC TGA AGG ATC CCT CAT TC-3' (SEQ ID NO:2). These oligonucleotide sequences were derived from the CAV

DNA sequence described by Noteborn et al, Journal of Virology 65, 3131-39 (1991). The template for amplification of probe by PCR was DNA extracted from MDCC-MSB1 cells using proteinase K (1 mg ml¹ proteinase K, 1 mM EDTA, 10 mM Tris, pH 8, 1% SDS) for 2 h at 37°C, followed by extraction with phenohisoamyl alcohol (24:1), and precipitation with ethanol at -20°C. The PCR was carried out as described (Novak & Ragland, Molecular and Cellular Probes 11, 135-41 (1997)). Amplified DNA was extracted from the PCR reaction mixture following the same procedure described for extraction of DNA from CAV-infected cells, omitting incubation with proteinase K. The amplified fragments of CAV DNA were labelled with biotin- 11-dUTP (Boehringer Mannheim, Indianapolis, IN, USA) by the nick translation method (Maniatis et al, MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, (1982)). Unincorporated nucleotides were removed by gel filtration using Quick Spin Columns (Boehringer Mannheim). The integrity of PCR product and success of the labelling procedure were confirmed by dot-blot hybridization (Maniatis et al. 1982) with known CAV DNA template using uninfected cell DNA as negative control. Additional tests for specificity were described earlier (Novak & Ragland,

1997).

Competitive Hybridization

A schematic diagram of the test appears in Fig. 2. The amount of capture probe immobilized onto nitrocellulose discs was set at

150 ng CAV DNA, measured by ratio of absorbence at 260 to 280 nm. The probe was immobilized in relaxed form. To determine the amount of labelled (biotinylated) probe to saturate the capture probe, increasing amounts from 1 to 200 ng were added to the capture probe in microtiter plate wells, followed with blocking reagent. Wells were rinsed, avidin- biotin-alkaline phosphatase complex added, and the wells rinsed again. Substrate was added and product allowed to form at room temperature for 30 min when the discs were removed. Amount of product formed was measured with a plate reader at 405 nm. The conditions were as described below for the derived test. Linearity of competition was determined by addition of increasing amounts of relaxed CAV DNA from

1 to 120 ng to the wells containing discs. The wells were incubated at 42°C overnight, and then the discs were washed. Blocking solution was added to the discs in wells, discs rinsed, and labelled probe was added, followed by substrate and colour development and measurement as described.

In the derived test, nitrocellulose discs with 150 ng of immobilized unlabelled CAV probe were placed in microtiter plate wells (flat bottom, 96 well plates) and prehybridized at 42°C for 4 h in a solution composed of 5x SSPE (0.9 M NaCl, 50 mM NaH,PO₄, 5 mM EDTA, pH 7.7), 5 x Denhardt's solution, 100 mg ml¹ denatured sonicated salmon sperm DNA and 0.1% (v/v) SDS. Hybridization with hybridization cocktail containing 45% formamide, 5x SSC, lx Denhardt's solution, 20 mM NaH₂P0₄, pH 6.5, 0J mg ml^"1 freshly denatured sheared herring sperm DNA, 5% dextran sulphate, and 1 mg DNA extracted from the sample (buffy coat) by proteinase K, phenol-chloroform-isoamyl alcohol extraction, was done overnight at 42°C.

Positive control was DNA extracted from CAV-infected MDCC-MSB1 cells, and negative control was DNA extracted from uninfected MDCC-MSB1 cells. Hybridization cocktail was removed from the wells, and replaced with hybridization cocktail containing 0.2 mg ml^"1 denatured biotinylated probe. Hybridization was allowed for 16 h at 42°C. Following removal of hybridization cocktail, wells containing nitrocellulose discs were rinsed several times in 2x SSC, 0.1% SDS, and washed in the same solution three times for 5 min at room temperature. Microtiter plate wells were then washed in three changes of 0J5x SSC,

01% SDS for 5 min each at room temperature, followed by three washes in the same solution for 30 min at 60° C. Prior to incubation in avidin- biotin-alkaline phosphatase complex, the nitrocellulose discs were incubated in blocking buffer (1% BSA in Tris-HCl, pH 7.5, 0.1 M NaCl, 5 mM MgCl₂, 0.25% Brij, pH 7.5) and then incubated with 100 ml of an avidin-biotin-alkaline phosphatase complex at room temperature for 30 min. The avidin-biotin-alkaline phosphatase complex was freshly prepared by combining 40 ml Avidin DN (Vector Laboratories, Burlingame, CA, USA), 5 ml of biotinylated alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN, USA) and 11 ml 1% BSA in TS Brij, pH 7.5. Wells were rinsed three times in TS Brij, pH 7.5, for 5 min at room temperature, and once in TS Brij, pH 9.5 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCtø. To each well containing a nitrocellulose disc was added 100 ml of substrate, p-nitrophenyl phosphate as supplied by Sigma Chemical (St. Louis, MO, USA), and colour was developed for 30 min at room temperature. Nitrocellulose discs were removed and absorbence measured at 405 nm with a microplate reader Sigma Chemical (St. Louis, MO, USA).

Detection Dot-blot assays

Extracts of buffy coats were assayed using biotinylated probe as described by Noteborn et al, Avian Pathology 21, 107-18 (1992). Hybridization was detected by binding of avidin-biotin-alkaline phosphatase complex, followed with colour development by incubation in reagents as described by McGadey (McGadey, Histochimie 23, 180-84

(1970)).

Probing blood smears

Blood smears were probed as described (Novak & Ragland, Molecular and Cellular Probes, 11, 135-41 (1997)). Blood smears from known CAV-positive and known CAV-negative chickens, confirmed by immunofluorescence assay for antibody (McNulty & Allan, RECENT ADVANCES IN VIRUS DIAGNOSIS (McNulty and McFerran eds). The Hague, Netherlands: Martinus Nijhoff (1984) were purchased from SPAFAS, Inc. (Storrs, CT, USA).

Virus isolation

Virus isolation from plasma, buffy coat, and erythrocytes was performed on MDCC-MSB1 cells as described by Yuasa et al, National Institute of Animal Health Quarterly, Japan 23, 75-5 (1983) using 7 transfers in 24 well tissue culture plates. Inocula were 0.25 ml each of plasma, buffy coat, and erythrocytes. Positive control was supernatant culture fluid from CAV-infected MDCC-MSBl cells that had been frozen and thawed three times to release virus, and then clarified by low speed centrifugation. Negative control was supernatant culture fluid obtained from uninfected MDCC-MSBl cells in the same way as for positive control.

Competitive ELISA

A CAV antibody test from IDEXX Laboratories (Westbrook, ME, USA) was used according to manufacturer's instructions.

Haematocrit

Freshly collected heart blood was placed on a plastic Petri dish and drawn into capillary haematocrit tubes lacking anticoagulant on days 0, 15, and 17, and subjected to centrifugation at 12,000 g for 5 minutes.

Adventitial infection All serum samples were tested by ELISA using commercial kits (IDEXX) for antibodies to Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, and reo virus.

Biometrics The Shapiro-Wilk W test for normal distribution of absorbence values in the MPT was done with the JMP program (SAS Institute, Inc., Cary, NC, USA). Correlation (r) analyses of integral data were done with the JMP program. Viral titers, excluding samples with zero titer, were transformed to log 10 for correlation analysis. Correlation analyses of nominal data (tests scored as positive or negative) were done by the method of Ives and Gibbons, A correlation measure for nominal data. American Statistician 21, 16-7 (1967), and statistical inferences determined by log likelihood ratio using the JMP program. Sample size to detect an infected flock (one-tailed test) was calculated for A=0.025, B=0.10, and D=0.44 (2 SD less than the mean of uninfected chickens) using the variance in the infected group (Zar, BIOSTATISTICAL ANALYSIS, 2nd edn. p.108 Englewood Cliffs, New Jersey: Prentice Hall, Inc. (1984)).

Sensitivity and specificity were measured according to Denson, Preventative medicine and public health; EPIDEMIOLOGY (Startwell, P.E., ed) p. 1-19, New York: Appleton-Century Crofts (1965). Youden Index for rating diagnostic tests. Cancer 3, p. 32-5 (1950) incorporated sensitivity and specificity in one calculation to derive a J index for which an index of 0 is a worthless test, and an index of 1 is obtained only when both sensitivity and specificity have values of 1. Although it places equal weight on both characteristics, it does provide a way of comparing performance of tests. Statistical inferences of J indices were done with Student's /test.

Results

Amount of labeled probe to saturated capture probe was determined to be 120 ng. Competition by CAV DNA was linear from 1 to 100 ng. (Figure 3)

Positive controls were positive for each test, and negative controls were negative for each test for CAV. None of the uninfected chickens was positive by any test.

Competition by CAV DNA in the MPT followed a quadratic relationship. It was linearized by log transformation from 5 to 100 ng (Fig. 1). Competition by 1 ng was outside the log-linear range. Slopes among the standard curves, and among the sample curves, were not all the same (Table 1 and Fig. 4), however slopes of standard and sample curves done at the same time were not different. Except for test 3, the slopes appeared to be members of a general relationship which has been depicted in Fig. 5.

Table 1

*Estimate ± standard error

The absorbence values in the MPT for uninfected chickens were normally distributed, and their mean absorbence was 1.23 with a standard deviation (SD) of 0.22, a coefficient of variation of 0.179 (data in Table 2 below). The variance was 0.4876 OD units². Positive MPT was arbitrarily set at 2 SD less than the mean absorbence of controls (ca. 95%o CI), viz. 0.79 OD units. None of the uninfected chickens was positive by this criterion.

Table 2

Competitive hybridization in microtiter plates for chicken anaemia virus nucleic acid in extracts of buffy coats from chickens free of chicken anaemia virus.

a Each value is for a different chicken measured in duplicate.

Results of tests on infected chickens have been recorded in Table 3 below. The mean absorbence was 0.60, SD 0J2, and variance 0.0466 OD units². As expected, virus isolation was the most reliable method of confirming presence of CAV. Whereas virus was not always recovered from plasma and erythrocytes, it was recovered from all buffy coats from the second through the 28th day. Among infected chickens, virus was isolated from 100% of buffy coats, 85%) of plasma samples, and 82% of erythrocytes. The MPT detected 72% of infected birds, in situ hybridization (ISH) 69%, dot-blots 67%, and ELISA 36%. Had the cutoff point for MPT been set at 1 SD, MPT would have detected all infected birds, but there would have been 6 false positives (14%) among uninfected birds. One infected chicken on day 7, positive by virus isolation, was not detected by MPT, ISH, dot-blot, or ELISA.

Correlation of birds positive by MPT with ISH was 0.44 (p=0.0005), with dot-blots 0.38 (p=0.08), with ELISA 0.08 (ρ=0.14), and with viral isolation from buffy coat 0.44 (p=0.0000). Correlation of absorbence values in the MPT with viral titers were 0.90 with plasma (p=0.0000), 0.69 with buffy coat (p=0.0000), and 0.56 with erythrocytes (p=0.0008).

Antibody for CAV was detected by ELISA beginning the seventh day. The ELISA detected only 56% of infected birds after appearance of antibody on the seventh day. From day seven on, ELISA did not detect CAV in 11 of 24 chickens that were positive by MPT, and in 8 of 22 chickens that were positive by dot-blot.

Sensitivity and specificity of the MPT were 0J8 and 1.00, respectively. For ISH, they were 0.72 and 1.00, and for the dot-blot test, they were 0J5 and 1.00. For virus isolation, sensitivity was 1.00 for buffy coat, 0.85 for erythrocytes, 0.75 for plasma, and specificity for all three was 1.00.

The J indices and standard errors for the MPT were 0.718 0.072 and 0.857 0.057 when cutoff values were 2 and 1 SD less than the mean absorbance of controls, respectively. The ISH had a j index of

0.718 0.072, dot-blots 0.667 0.075, and ELISA 0.385 0.078 (cutoff value at 2 SD less than the mean absorbency of controls). When the MPT was compared with dot-blots, it was significantly different (pθ.05) only when the cutoff was 1.0 SD less than the mean absorbency of controls. The MPT was significantly different from ELISA (pO.Ol) even when the most conservative cutoff (2 SD less than mean absorbency) was used. The MPT and ISH were not statistically different at either cutoff level.

The sample size necessary for a 90% chance of detecting a positive chicken in the infected group at the 0.05 level of significance was three chickens.

Haematocrit values were normal at the beginning of the experiment, and were less than 27% on days 15 and 17, confirming presence of moderate anaemia. All serum samples were negative for adventitial infections throughout the course of the experiment.

Table 3 Comparison of Several Methods for Detecting Infection 'with Chicken

Anaemia Virus Chickens were infected by intracoelomic injection at one day of age, and samples taken from three chickens sequentially thereafter for virus isolation, enzyme-linked immunosorbent assay (ELISA for antibody to the virus) molecular hybridization of buffy coats on dot-blots, in situ hybridization on blood smears (ISH), and a competitive DNA hybridization assay in microtiter plates (microtiter plate test).

Chickens were infected by intracoelomic injection at one day of age, and samples taken from three chickens sequentially thereafter for virus isolation, enzyme-linked immunosorbent assay (ELSIA for antibody to the virus), molecular hybridization of buffy coats on dot-blots, in situ hybridzation on blood smears (ISH), and a competitve DNA hybridzation assay in microtitre plates (microtitre plate test).

Positive arbitrarily set two standard deviations less than mean absorbance of uninfected chickens, ±0.79. No sample.

Conclusion

Virus isolation continues to be the most reliable method of confirming CAV infection. Whereas virus was present in plasma and erythrocytes, buffy coat was the most reliable source and the one used for a reference standard by us. This is the first report of CAV in erythrocytes. Although it has been reported that serum is not infectious after 14 days (von Blow & Schat, Chicken infectious anemia. In Diseases of Poultry, 10th edn. (Calnek, et al. eds) p. 739-56 Ames, Iowa: Iowa State University Press (1997)), we isolated virus from plasma of some chickens through the 28th day. Buffy coat has been reported to be infectious for as long as 14 days (von Blow & Schat, Chicken infectious anemia; DISEASES OF POULTRY, 10th edn. (Calnek, et al. eds) p. 739-56 Ames, Iowa: Iowa State University Press (1997)), and we recovered virus from buffy coat through the 28th day. Poor correlation of MPT with virus isolation from buffy coat was a result of setting the absorbence level for a positive sample at 2 SD less than the mean of controls. The correlation probably would have been stronger had TCID₅₀ titers been determined, as they would have been more accurate than the non-parametric method we used. Further refinement of the parameters for the MPT should improve the correlation.

It would have been better at a less restrained level if false positives could be accepted, e.g. r=l if the cutoff level is set at 1 SD less than the mean of controls. For the purpose of this test, where a group of birds from a flock will be sampled to detect presence of virus in the flock, false positive results cannot, or should not, be tolerated. At least three chickens must be sampled from an infected flock. However, this calculation was based on chickens that were infected for one to 28 days. Although from clinical experience the majority of a flock will be infected, the percentage of infected birds in a flock when it is sampled cannot be known. For this reason, a larger sample size, probably five to seven chickens, should be sampled as an added measure of confidence. In tests for other viruses, a balance between false positive and negative results may be acceptable, depending on the use of the test, and for some tests false negatives may not be acceptable while false positives may be. The least reliable method in this study was ELISA, even after antibody response had developed. Dot-blot, MPT, and ISH had about the same reliability although they were not concordant. The cutoff point for the MPT was selected arbitrarily at 2 SD less than the mean of controls which theoretically allows 2.5% of observations to fall below the cutoff. The number of observations below the cutoff probably is less for two reasons. Although the distribution was normal, it was skewed toward higher values, indicating fewer observations would occur below the cutoff point. Furthermore, one sample estimates (one experiment) overestimate observations that would fall outside a 95% CI. If the cutoff point had been 1.5 SD less, sensitivity would have been 0.81 and specificity 0.93. If it had been one SD less, sensitivity would have been 0.95 and specificity 0.89.

The MPT and the ELISA should be used in combination as they address two different aspects, presence of virus, and immune response to prior or present infection. Natural infection of breeder flocks has been used, and live vaccines in Europe, to reduce losses from CAV.

Nevertheless, broiler chickens hatched from these flocks, while protected by maternal antibody during their early weeks, will become infected.

Whereas losses in these chickens are not as dramatic, they are significant (McNulty et al, Avian Diseases 35 263-8 (1991)). As non-viable vaccines become available, these losses will be reduced further. The

MPT and the ELISA are complementary; MPT to diagnose infection, and

ELISA to monitor strength of immunity from vaccination.

The MPT has potential for measuring amount of CAV DNA from a standard curve, and this may be useful in determining virus load in a sample.

Example 6

Competitive Nucleic Acid Hybridization Assay

Method

Preparation of samples from animals

Pathogen-free chickens are used and housed in conventional batteries kept in rooms supplied with filtered air under positive pressure. Chickens infected with CAV are kept in one room, and uninfected chickens are kept in another room. Status of the chickens is confirmed and monitored by in situ hybridization on blood smears.

Blood samples (500 μl) from chickens infected with CAV and anticoagulated with EDTA, are mixed with 1 ml of 37.5 mM NaCl and centrifuged 800 x g for 5 minutes at 4°C to sediment cells.

Supernatant solution is discarded and the pellet is washed several times with the salt solution to remove most of the hemoglobin. Pelleted lymphocytes and erythrocyte nuclei is suspended in 200 μl of 1 mM EDTA, 10 mM Tris, 1% SDS, and 1 mg/ml proteinase K, adjusted to pH 8. Proteolytic digestion by proteinase proceeds for 2 hours at 5CCC. 200 μl of hybridization cocktail (as described above) is added and the mixture is placed in a boiling water bath for 10 minutes to relax the DNA.

Preparation and labeling of nucleic acid probes Hybridization probes are prepared according to the methods described in the above Examples.

Assay

Aliquots of as much as 200 μl is assayed in the derived test. First, blood from uninfected chickens is spiked with known amounts of CAV DNA. Then a range of volumes from infected chickens is tested.

Detection

A range finding assays are done only to establish the appropriate volume to be assayed. Routine assays use the determined volume. When assays are done on blood samples, wells containing known amounts of CAV DNA are used as positive controls, and wells with foreign DNA are used as negative controls.

Blood samples are collected weekly from infected and uninfected chickens for three to four months and assayed. Results are compared with presence of antibody Q LISA) and presence of infectious virus (cell culture) using paired serum and blood samples from the same chickens.

Simple or multiple correlation, whichever is appropriate for the data, is used when comparing methods. Confidence intervals are set at 95%.

Table 4

Chicken anaemia virus titers in plasma, buffy coat, and erythrocytes of infected chickens compared with competitive nucleic acid hybridization for virus in microtiter plate tests (MPT).

First cell passage having no viable cells. Viable cells after 7 passages. No sample.

Example 7

Induction of IFN mRNA Method Preparation of samples

Specific-pathogen-free eggs were purchased from Charles River Farms Hungary (Budapest), hatched in a conventional egg incubator, and the chickens raised in a conventional wire battery. The chickens were provided starter ration and water ad libitum. Serum samples were collected at the conclusion of each experiment and tested for most common poultry diseases to confirm the chickens had been maintained free of infection.

Since RNA is highly susceptible to degradation by ribonucleases, and blood is rich in ribonucleases, prevention of mRNA degradation is an important consideration in sample preparation. Catrimox-14, a cationic detergent, preserves mRNA for 14 days from 4°C to 37°C. making it an ideal blood preservative. Since the same amount of mRNA is induced from one day to 4 weeks of age, the oldest age tested, abundance or message at different ages can be compared.

Anticoagulated blood was taken for total white blood cell counts in a haemocytometer using Natt and Herrick's stain (Campbell,

1988). Blood smears were made and stained with Giemsa stain for differential counts of mononucleated cells and granulocytes. Duplicate Newcastle disease haemagglutination inhibition micro assays were done using two-fold dilution of serum and plasma samples (Swayne et al., 1998).

Preparation and labeling of nucleic acid probes

Plasmids containing IFNα and γ DNA were obtained from Drs. M. J. Sekellik and P. I. Marcus (1994). and J. W. Lowenthal (Digby and Lowenthal, 1995), respectively. Competent E. coli strain DH5α was transformed with either SPORTI plasmid containing chicken IFNα DNA or pUC18 plasmid containing chicken IFNγ DNA using the CaCl₂ method (Sambrook et al., 1989). Transformed cells were grown in LB medium and DNA extracted from overnight cultures using QIAGΕN Plasmid Mini Kit (Hilden, Germany) according to manufacturer's instructions. The amount of plasmid DNA was measured spectrophotometrically. Capture probe was immobilized onto nitrocellulose discs. Detection probe was labeled with biotin by nick translation according to instructions provided with BioNick Labeling System (GIBCOBRL, Life Technologies, Inc., Berlin, Germany).

Unincorporated nucleotides were removed by spun-column chromatography using Sephadex G-50 (Sigma, Diesenhofen, Germany). Sequence analysis was done to confirm the probes contained the correct nucleic acid sequence for the respective IFN.

Preparation of RNA

Total RNA extraction from blood using proteinase K-phenol method

Three-tenths milliliter of heparinized blood was centrifuged 250 g at 4°C for 30 min. Buffy coat layer together with some overlying plasma and underlying erythrocyte (to recover all white blood cells) was pipetted into a fresh tube. The white blood cells were mixed with 500 μl of ice-cold extraction buffer (0.15 M NaCl, 10 mM. Tris-HCl, pH 8.5, 0.5% IGEPA1, 1 mM dithiothreitol, 20 mM vanadyl- ribonucleoside complexes) and vortexed briefly. One hundred and sixty micrograms of proteinase K in proteinase K buffer (0J M Tris-acetate, 0.3 M sodium acetate, 50 mM EDTA, pH 7.5, 2% w/v SDS) was added to each sample. Samples were incubated at 37°C overnight. An equal amount of buffer-saturated phenol and 320 μl of 3 M sodium acetate were added, and extraction was allowed at room temperature for 5 min. Samples were centrifuged at 14,000 g for 2 min to separate organic and aqueous phases. The aqueous phase was transferred to a new tube and extracted with phenol:chloroform:isoamyl alcohol (25:24:1) at room temperature for 5 min. Phases were separated by centrifugation at 14,000 g for 2 min. The upper phase was transferred to a new tube and extracted with an equal volume of chlorofornrisoamyl alcohol (24:1, v/v) for 5 min at room temperature. The aqueous phase was transferred to a new tube and 2.5 volumes of ice-cold absolute ethanol were added to each sample. Total RNA was precipitated on dry ice for 30 min and centrifuged at 14,000 g for 10 min. at 4°C. Pellets were washed in 80% ethanol and resuspended in 10 mM Tris-HCl and ImM EDTA, pH 8.0, (TE) buffer, or stored at -80°C until assayed.

Total RNA extraction from Catrimox-14 preserved blood

Heart blood was added to Catrimox-14 in microcentrifuge tubes at a ratio of 0.1ml blood to 0.5ml Catrimox-14 (Machfarlane and Dahle, 1993; Schmidt et. al., 1995). Total RNA was extracted using the acid guanidinium-phenol-chloroform method. Comparable volumes of blood were preserved either in proteinase K or Catrimox-14 at 4°C, room temperature, and 37°C for I hr, 1 and 2 week, when they were assayed by the appropriate method. This was done to confirm the same amount of mRNA was obtained by either method. Extracted RNA was stored at -

20°C until assayed. Catrimox-preserved samples now are kept at room temperature. Table 5

Amount of interferon (IFN) mRNA in aliquots of peripheral blood preserved in proteinase K or Catrimox-14 at 4C, Room Temperature (RT) or 37°C, and assayed I hr, 7 and 14 days later.

Competitive Hybridization

Blocking competition assays were done in microtitre plates using the same method described in previous examples and Figure 1. The amount of capture probe immobilized onto nitrocellulose discs was determined by titrating IFNα and γ DNA with labeled probe.

Probe was denatured by heating at 100°C for 10 min, and addition of an equal volume of 20x SSC. Denatured probe, 100 ng per disc, was immobilized by baking at 80 °C under vacuum for one hour. Discs were briefly rehydrated with DEPC-treated water, and placed in microtitre plate wells together with prehybridization cocktail containing 5x SSPE (0.9 M NaCl, 50 MM NaH2F04, 5 mM EDTA, pH 7.7), 5x Denhardt's solution, 100 mg/rnl denatured sonicated salmon sperm DNA, 1 μg/ml DNA of the corresponding non-transformed plasmid and 0.1% (v/v) SDS. Prehybridization was allowed for 4 hr at 42°C. Prehybridization cocktail was replaced with hybridization cocktail containing 45% formamide, 5x SSC, IX Denhardt's solution, 100 mg/ml freshly denatured sheared herring sperm DNA, 5% dextran sulphate, and 5μl of each extracted, denatured total RNA preparation. Positive control was DNA for chicken IFNα and γ, respectively. Negative control for IFNα was IFNγ DNA, and for IFNγ, it was IFNα DNA. Hybridization was allowed overnight at 4°C. Hybridization cocktail was removed and replaced with hybridization cocktail containing denatured, labeled probe excess. Hybridization was allowed for 16 hr at 42°C. Following removal of hybridization cocktail, wells containing nitrocellulose discs were rinsed several times in 2X SSC, 0. 1% SDS, and washed in the same solution three times for 5 min at room temperature. Microtitre plate wells then were washed in three changes of 0J5X SSC, 0.1 % SDS for 5 min each at room temperature, followed by three washes in the same solution for 30 min at 60°C. Prior to incubation in avidin-biotin-alkaline phosphatase complex, the nitrocellulose discs were incubated in blocking buffer (1% BSA in Tris-HCl, pH 7.5, 0.1 M NaCl, 5 MM MgCl₂, 0.25% Brij, pH 7.5) and then incubated with 100 μl of an avidin-biotin-alkaline phosphatase complex at room temperature for 10 min. The avidin-biotin- alkaline phosphatase complex was freshly prepared by combining 40μl

Avidin DN (Vector Laboratories, Burlingame, CA, USA), 5μl of biotinylated alkaline phosphatase (Boehringer Mannheim, Germany) and 11 ml 1% BSA in TS Brij, pH 7.5. Wells were rinsed three times with TS Brij, pH 7.5, for 5 min at room temperature, and once with alkaline phosphatase buffer, pH 9.6 (2.5 mM MgC12. 1 M diethanolamine). To each well containing a nitrocellulose disc was added lOOμl of substrate, p-nitrophenyl phosphate, as supplied by Sigma Chemical (St. Louis, MO, USA), and color was developed in the dark for 30 min at room temperature. Nitrocellulose discs were removed and absorbence measured at 405 nm with a microtitre plate reader.

Results

In one experiment, at 3 weeks of age, chickens were challenged by intracoelomic (IC) injection of iNDV (Pestikal vaccine, Pliva d.d., Zagreb), while a control group was not. Heparinized blood was collected by heart puncture immediately before vaccination, at 4, 24, 48,

72, and 168 hours after challenge. The lymphocytes were collected, total

RNA extracted from them, and IFN mRNA assayed as described above.

A spline curve applied to the data indicated maximum abundance of message around 6 hours, followed by a decline, a secondary rise, and return to baseline (no measurable message) by 7 days. The JMP statistics I program (SAS institute, Inc., Cary, NC) was used for various analyses as reported in the tables and figures. Statistically significant inference was accepted at p<0.05.

In a second experiment, samples were collected at 0, 2, 4, 6, 8, 12, 18, 24, 30, 36, 42m 48, 54, 60, 66, 72, 78, 90, 96, 102, 120, 144, and 168 hr after challenge with iNDV. Blood samples were collected in Catrimox-14 and total RNA extracted by the acid guanidinium isothiocyanatephenol-chloroform method. Maximum abundance was reached at 4 hr, remained high for 3 days, after which it declined to baseline on the fifth day.

In a third experiment, heart blood was collected 4 hr after IC challenge with iNDV at 1, 2, 5, 7, 14, 21, and 28 days of age (Table 6 below). Samples were collected also from non-challenged chickens and it was determined that the level of induction was the same at all ages (p<0.05).

Table 6

Abundance of mRNA for interferons induced 4 hr after challenge with inactivated Newcastle disease virus at various ages.

eren , p> . .

In a fourth experiment, chickens were injected IC with 0, 2.5 and 5 mg CY from 14 through 16 days of age. Six chickens from each group were challenged IC with iNDV on the 16th day. Heart blood was collected from them and 6 naive chickens 4 hr later for assay of IFN mRNA. Serum samples were collected (wing vein) for viral antibody (HI) one week later. The same protocol was repeated 1 and 2 weeks later. The mRNA assays were done as described. Newcastle disease virus HI titres were done by conventional micro assay as describes above. The high dose of CY initially suppressed transcription of both IFN mRNA, the chickens recovering several weeks later (Tables 7 and 8 below). Correlation of IFNα mRNA with antibody responses was 0.93, and correlation of IFN/ mRNA with antibody responses was 0J1.

Table 7

Abundance of interferon mRNA in chickens treated with 1, 2.5 and 5 mg cyclophosphamide (CY) from 14 through 16 days of age, and different groups challenged IC with inactivated Newcastle disease virus on the 16th, 23rd and 30th days, and HI titres for Newcastle disease virus. Naive group was challenged on day 30.

tests for HI titres.

Table 8

Abundance of interferon mRNA in blood of chickens treated with 0 and 4 mg cyclophosphamide (CY) from 18 through 20 days of age, and challenged with inactivated Newcastle disease virus on days 20, 27, 34, 41 and 55. Blood samples were taken 4 hr after challenge. A naive group was not challenged.

alphabetic superscripts are different at p< 0.05 by log likelihood tests.

In a fifth experiment, chickens were treated with 0 and 4 mg CY and half of the untreated chickens were not challenged with iNDV. Anticoagulated blood samples were also collected and used to count total white blood cells in a haemocytometer. Blood smears also were made and differential counts made of mononuclear lymphocytes (thrombocytes were excluded) and granulocytes. The suppression of IFN mRNA transcription was again observed, followed by recovery. Treatment with CY did not cause lymphopanemia, demonstrating that the decrease in IFN mRNA resulted from a molecular interference with transcription, not depletion of number of circulating leukocytes (Table 9 below). Table 9

White blood cells (WBC), mononuclear cells (MNC) and granulocytes (Grans) in heart blood of chickens injected intracoelomically with 0 and 4 mg cyclophosphamide for three days, and challenged with inactivated Newcastle disease virus the third day, 1, 2, 3 and 5 weeks later. Blood samples were taken 4 hr after challenge. A naive group was not challenged.

treatment.

²Means + std. dev. (no. of observations), means in each row are not different, p>0.05 by Kruskal- Wallace test.

In a sixth experiment, four-week-old, specific-pathogen- free chickens reared in isolator units supplied with filtered air under positive pressure were challenged IC with 0.4 ml CAV DelRoss strain (10⁷ TCID₅₀/01 ml). Uninfected chickens from the same hatch were housed together in separate units. Chickens were infected at 4 weeks because this protocol has been shown to cause infection without anemia, the purpose of the experiment being to as-say immune suppression in inapparent, subacute infection. One, two and three weeks later a group of infected and a group of uninfected chickens were challenged IC with iNDV. Chickens were used only once. Heart blood was collected 4 hr later from them and from 3 naive chickens. Two tenths of a milliliter was preserved in 1 ml Catrimox-14, and 0.2 ml allowed to clot for collection of serum to be used in ELISA for CAV. Blood was also collected from wing veins into EDTA for hematocrit measurement and WBC counts. The Catrimox-14 samples were equally divided, DNA was extracted for assaying CAV by competitive hybridization from half (0.1 ml), and RNA was extracted from the other half for assaying IFNα (Table 10 below) and IFNγ (Table 11 below) mRNA. Table 10

Abundance oflFN mRNA 4 hr after challenge with inactivated Newcastle disease virus (iNDV) in chickens infected and not infected with chicken anaemia virus (CA V) at 4 weeks of age.

a a ues n co umns with di erent a p abetic superscripts are different at p<0.05 by rank sums tests.

Table 11

Abundance oflFNγmRNA 4 hr after challenge with inactivated Newcastle disease virus (iNDV) in chickens infected and not infected with chicken anaemia virus (CAV) at 4 weeks of age.

Age when challenged with virus reatment AV iNDV 5wk 6wk 7wk

0.7338 ± 0.0628(3)¹ 0.8420 ± 0.0232(3)¹ 0.8563 ± 0.0230(3)¹

0.0714 ± 0.013 l(5)b 0.0971 ± 0.0147(5)b 0.3097 ± 0.0553(5)

^{+ +} I 0.5023 ± 0.0475(7)^c | 0J031 + 0.0578(7)^c | 0.7276 ± 0.0744(8)c a Values in columns with different alphabetic superscripts are ditterent at p< 0.05 by rank sums tests.

Results

All infected chickens in experiment 6 were positive for CAV by ELISA and competitive nucleic acid hybridization. None of the other chickens was positive. Haematocrits and white blood cell counts were within normal ranges and not different among all groups, demonstrating inapparent infection. Transcription for both IFN was induced by iNDV, while no measurable IFN mRNA was found in naive chickens. Induction of IFNα mRNA, the type most associated with viral immunity, by iNDV was totally suppressed by CAV, while transcription of IFNγ mRNA was less markedly but significantly suppressed (65% to 80%)) by CAV. Thus, CAV not only suppresses immunity by interfering with IFN, it does so at the transcriptional level.

The induction of chicken mRNA for IFNα and γ can be measured in microtitre plates with the described test. Induction is the same regardless of age of the chicken. This means chickens can be tested at any age and data easily compared. We have chosen to collect samples 4 hr after induction, but the delay probably can be reduced to 3 or even 2 hours. Kinetics of induction was consistent with known parameters for vertebrates. Decrease in mRNA by cyclophosphamide treatment correlated with dose, and with HI antibody titres for Newcastle disease virus one week after challenge. The test has been successfully applied to inapparent, subacute infection with chicken anemia virus, known to be immunosuppressive. Interference with transcription of IFNα mRNA was 100% while interference with transcription of IFNγ mRNA was 65-80%.

The coefficient of variation is quite small and only 5 SPF chickens per group are needed for significant inference at p< 0.05. The data have been recorded on basis of absorbence. Using cDNA standards for both IFN mRNAs for standard curves, the absorbence values can be converted to nanograms or μmoles of each IFN mRNA induced, i.e. a quantitative measure. Thus, immune suppression can be measured with the test, and the abundance of IFN mRNA induced correlates with antibody responses.

Example 8 Quantification of IFN mRNA in a sample

Method Production of IFN standards

The template used in PCR was cDNA produced by reverse transcription of total RNA extracted from the spleen of a chicken that had been challenged with inactivated Newcastle disease virus 4 hr earlier.

Spleen cells were separated by pressing the tissue through a screen mesh, and the lymphocytes were separated from the cell suspension by centrifugation over Ficoll-Hypaque. Total RNA was extracted from the lymphocytes with acid guanidinium-phenol-chloroform method. It was used to produce cDNA by reverse transcription with Enhanced Avian RT-

PCR kit from Sigma, following manufacturer's instructions. Primers used in PCR were: 5'-AGA AGA CAT AAC TAT TAG AA-3' (SEQ ID NO:9) and 5'-TTA GCA ATT GCA TCT CCT CT-3' (SEQ ID NO: 10). Cycling conditions were 94°C for 2 min, 30 cycles of 94°C for 1 min, 50°C for 2 min, 72°C for 2 min, and finally 7 min at 72°C. The resulting IFNγ standard PCR product was 3412 base pairs. The same template for IFNγ standard was used with the primers: 5'-ATG GCT GTG CCT GCA AGC CCA-3' (SEQ ID NO: 11) and 5'-CTA AGT GCG CGT GTT GCC TGT-3' ((SEQ ID NO: 12). Cycling conditions were 2 min at 94°C, 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and finally 72°C for 7 min. The resulting IFNα standard PCR product was 582 base pairs.

Preparation of samples

A 3-week-old specific-pathogen-free chicken was challenged intracoelomically (IC) with inactivated Newcastle disease virus as described above. Four hours later, 0.2 ml heart blood was preserved in 1 ml Catrimox-14, and total RNA extracted using the acid guanidinium-phenol-chloroform method.

In search of a test that could be used in clinical avian medicine, i.e. in chicken houses, it was important to find a preservative for nucleic acids that was not hazardous and would preserve the mRNA until it could be assayed in a laboratory. Preservation with Catrimox-14 and proteinase K were compared. The amount of mRNA detected with Catrimox-14 or with proteinase K was the same for samples kept at 4°C to 37°C for at least 2 weeks. Catrimox-14 is a cationic detergent that is not hazardous or toxic, and special conditions will not be required for transporting samples. Consequently, Catrimox-14 is an ideal preservative for samples collected in the field.

Table 12

Comparision of the two preservation and extraction methods

The two methods measured comparable amounts of mRNA (Table 5 above). Whole model analysis of the data was:

Competitive hybridization

The described competitive assay was done using dilutions of the prepared standards to compete with labeled probe for capture probe. Dilutions of the sample RNA were recorded as volume of heart blood.

Linearity of probe binding

Linearity of binding for the probes of SEQ ID NO:9-12 are shown in Fig. 6. The binding curves were quadratic, and they were linearized by log transformation.

Results

Standard curves were developed for the quantification of IFN mRNA (Fig. 7 and Fig. 8) using the same approach as for the quantification of CAV. In both figures, standard curves were on the left while sample dilutions were on the right. Broken lines are 95% confidence intervals. R squares are greater than 0.99 for all lines. Slope for IFNα mRNA standard was -0.928 and for the corresponding sample it was -0.870. Analysis of covariance for the two slopes confirmed the slopes were not different, p>0J8. Slope for IFNγ mRNA standard was -

0.824 and for the corresponding sample it was -0.783. Analysis of covariance for the two slopes confirmed the slopes were not different, p>0.85. Since the slopes for each message and its corresponding sample were not different, the regression equation for each standard can be used to calculate the abundance of its corresponding message in a sample. In the example, abundance would be recorded by volume of blood, but other references could be used, for example, per cell. The same method can be used to quantify any mRNA of interest providing slopes of standard and sample are not different. Similarly, the same method can be used to quantify any nucleic acid sequence of interest.

Kinetics of induction of IFN mRNA

Blood samples were collected 0, 4, 6, 24, 48, 96 and 144 hours after challenge in the first experiment. A spline curve applied to the data indicated maximal abundance of message around 4 hours, followed by a decline, a secondary rise, and return to baseline (no measurable message) by 7 days. The experiment was repeated, taking samples more frequently (0, 2, 4, 6, 8 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 78, 90, 102, 120, 144 and 168 hours) after challenge (Figs. 7 & 8). Maximal abundance of each message was again at 6 hours, after which it remained at high levels for 3 days, and then declined to baseline on the fifth day.

Message was not detected in unchallenged chickens.

Temporal induction of mRNA

Message was detected for both IFN in challenged chickens but not unchallenged chickens, the difference between challenged and unchallenged chickens being statistically significant (data not shown).

There were no differences in abundance of message for each of the IFN in challenged chickens by time.

Chickens immune suppressed with CY

In both experiments, 4-5 mg CY interfered with transcription for both IFN initially, and it lasted longer for α than for γ.

The high dose in the first experiment also depressed antibody responses, which correlated with abundance of mRNA (IFN-α at 0.93, and IFN-γ at 0.71). There were no differences for each category of white blood cells by treatment or time, so the data were pooled (Table 8 above).

Conclusions

The assay is a competitive blocking test as depicted in Figure 1. The labelled probe is in excess to saturate the capture probe. If complementary nucleic acid sequences are allowed to bind before labelled probe is applied, they will block binding of labelled probe proportional with the amount of capture probe blocked. Linearity of binding of probe to capture probe demonstrated there was no interference with their binding, such as can occur if capture probes are in too close proximity

((Chan et al, Biophysical 1, 69, 2243-2255 (1995)).

It further implied the competition assay would be linear, as was subsequently observed. Signal in the competition assay is inversely proportional with amount of the specific sequence in the test sample. Figure 6 demonstrates the binding of labeled probe to capture probe. This demonstrated that the assay was proportional to the blocking compound (either DNA or RNA). Whereas data reported here are absorbence values, using cDNA for IFN mRNA standards, we have found the slopes of curves for standard cDNA and sample RNA extracts are not different, indicating that in future we should be able to quantify on a molecular basis the amount of message in a sample.

Kinetics of induction was similar for both IFN. Data were subjected to best fit analysis. The rapid rise in abundance to near maximal levels in 4 hours, gave us an opportunity to collect samples 4 hours after challenge with iNDV. Samples can be collected from several farms in one day. Message probably can be detected as early as one hour but this would be too soon. Since we want to detect interference with transcription in immune compromised chickens, samples should be collected near normally peak levels. Since the same levels of message were induced in chickens from 1 to 28 days of age, tests made at different ages can be compared.

We could detect interference with transcription by CY, and the abundance of message was a predictive measure of immune status as ascertained by NDV HI titres. Cyclophosphamide has been used to bursectomise chickens by treating them with high doses during the first four days of life ((Rouse et al, Austral. J. Expt. Med. Sci., 52, 873-885

(1994)). We did not want to ablate immune responsiveness, only impair it, which was accomplished by the high doses of CY. In both experiments, the high dose severely interfered with transcription of both messages. In the first CY experiment, data indicated modest but significant interference (more so for α than for γ) at 2 weeks (30 days of age) for all doses of CY, whereas the lower doses did not interfere at earlier times. In the second experiment, interference with α appeared to be waning at 1 week, and the chickens had not fully recovered from the interference at 2 weeks, which would be concordant with the first experiment. Although there was not a significant difference for γ at 1 week, there may have been some interference, as there was for α, but it was obscured by the high standard deviation for the naive chickens. Clearly, there was no interference with transcription by the third week.

We were able to detect immune suppression by CY, but we did not know whether the suppression was result of spedfic effects on molecular events leading up to transcription or a reflection of leukopacnia. In either case, we were able to identify immune compromised chickens. The second CY experiment was done to determine if the decrease in IFN mRNA was due to leukopasnia. While CY treatment interfered with transcription, there was no difference in total white blood cells, mononuclear cells or granulocytes in the circulating blood (Table 9 above). Thus, the decrease in message resulted from interference with transcription.

The proposed test protocol to measure induction of IFN mRNA comprises several steps: • In a chicken house, collect chickens, label them, and draw 0. 1 ml blood into 0.5 ml Catrimox-14. Challenge the birds with an immunogen (iNDV). Place birds in a chicken crate for approximately 1-5 hours, preferably 2 or 4 hours. • Collect a second blood sample.

Release the chickens.

Transport "before" and "after" samples to laboratory (test takes 2 days).

Standard curves with cDNA can then be developed to assist in further diagnosis. Although there is a report that IFN levels are suppressed in subclinical infections of chicken anaemia virus, interference with transcription of message for IFN has not been reported. Thus, the test may be used to determine if any chicken viruses interfere with transcription for

IFN. Futhermore, as all immune suppressive viruses of humans appear to interfere with transcription for IFN, the chicken viruses probably do also.

Example 9

Kit for screening various groups of diseases in humans and animals A kit comprising various probes for the major pathogens in respiratory diseases could be used for preliminary testing for the presence of infectious agents. A positive result for a specific pathogen could be followed by a specific PCR based test involving mixtures of specific reagents in several different combinations, and a single organism can be diagnosed from the pattern obtained in the tests. Alternatively, each row in a microtitre plate could have capture probes for a different pathogen common to respiratory infections (or intestinal, urologic, etc. infections). Kits using either approach could be used to diagnose infections by genus, if the cause is among the common pathogens. If a common pathogen is not diagnosed, the cause may be a less frequent pathogen, even an emerging pathogen, thus the patient may warrant further study. If a common pathogen is detected, there may be no need for a species or strain diagnosis. However, such diagnosis could be done using the PCR based methods described herein.

Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

Claims

ClaimsWhat is claimed is:

1. A method of detecting target infectious agents in a sample, comprising: preparing a probe from a nucleic acid of the target infectious agent and immobilizing the probe on a matrix; obtaining the sample; incubating the sample with the first immobilized probe under conditions favorable for hybridization of the sample to the first immobilized probe; labeling the second probe; incubating the second labeled probe with the sample and first immobilized probe in an amount in excess of the first immobilized probe; and detecting how much of the second labeled probe has bound to the first immobilized probe wherein if the target infectious agent is present in the sample, the target infectious agent in the sample will compete with the second labeled probe for the first immobilized probe, thereby decreasing subsequent detection of the amount of the second labeled probe.

2. The method of claim 1 wherein the probe is derived from chicken anaemia virus.

3. The method of claim 1 wherein the probe is derived from HIV-1.

4. The method of claim 1 wherein the probe is derived from interferon.