US20050170341A1

US20050170341A1 - Methods for detecting Ehrlichia canis and Ehrlichia chaffensis in vertebrate and invertebrate hosts

Info

Publication number: US20050170341A1
Application number: US10/138,162
Authority: US
Inventors: Roger Stich; Yasuko Rikihisa
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-08-25
Filing date: 2002-05-02
Publication date: 2005-08-04
Also published as: US6432649B1

Abstract

Tools and methods for detecting the presence of E. canis and E. chaffeensis in a sample obtained from an animal are provided. The methods employ a polymerase chain reaction and primer sets that are based on the p30 gene of E. canis and the p28 gene of E. chaffeensis. The present invention also relates to the p30 and the p28 primer sets. Each p30 primer set comprises a first primer and the second primer, both of which are from 15 to 35 nucleotides in length. The first p30 primer comprises a sequence which is complementary to a consecutive sequence, within the following sequence: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30 primer comprises a sequence which is complementary to the inverse complement of a consecutive sequence contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2. The first p28 comprises a sequence which is complementary to a consecutive sequenc, within the following sequence: A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO. 3. The second p28 primercomprises a sequence which is complementary to the inverse complement of a consecutive sequencewithin one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO. 4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO. 5.

Description

BACKGROUND OF THE INVENTION

The Ehrlichiae are obligate intracellular bacteria found in circulating leukocytes of infected animals. Ehrlichia canis (E. canis) infects monocytes and causes ehrlichiosis in animals belonging to the family Canidae. E. canis is transmitted by the brown dog tick, Rhipicephalus sanguineus.
Canine monocytic ehrlichiosis (CME) consists of an acute and a chronic phase. The acute phase is characterized by fever, serous nasal and ocular discharges, anorexia, depression, and loss of weight. The chronic phase is characterized by severe pancytopenia, epistaxis, hematuria, blood in feces in addition to more severe clinical signs of the acute disease. If treated early during the course of the disease, dogs respond well to doxycycline. However, chronically infected dogs do not respond well to the antibiotic. Therefore, early diagnosis is very important for treating canine ehrlichiosis.
Human monocytic ehrlichiosis (HME) is a tick-borne, emerging infectious disease that is caused by the rickettsial pathogen, Ehrlichia chaffeensis (E. chaffeensis). The course of HME begins with an asymptomatic pre-patent period, followed by an acute phase where the vertebrate host suffers pyrexia, anorexia, weight loss, cytopenia and even death. Non-human hosts that survive the acute phase typically undergo partial recovery and suffer mild chronic infections, during which they could be persistent carriers that are capable of infecting tick vectors. Both dogs and white tailed deer are susceptible to infection with E. chaffeensis, and both of these hosts are suspected reservoirs of this pathogen. E. chaffeensis appears to be transmitted by Amblyomma. americanum, and appears to be endemic to the southern U.S. where this tick is indigenous. Symptoms of human monocytic ehrlichiosis (HME) are similar to those of canine monocytic ehrlichiosis (CME) that is also known as tropical canine pancytopenia. (Hildebrandt, P. K., D. L. Huxsoll, et al. (1973 (1973). Pathology of canine ehrlichiosis (tropical canine pancytopenia). Am J Vet Res 34(10): 1309-20.; Kuehn, N. F. and S. D. Gaunt (1985). Clinical and hematologic findings in canine ehrlichiosis. J Am Vet Med Assoc 186(4): 355-8.; Eng, T. R., J. R. Harkess, et al. (1990). Epidemiologic, clinical, and laboratory findings of human ehrlichiosis in the United States, 1988. Jama 264(17): 2251-8.; McDade, J. E. (1990). Ehrlichiosis—a disease of animals and humans. J Infect Dis 161(4): 609-17.) The etiologic agents of CME and HME, E. canis and E. chaffeensis, respectively, have been placed in the same genogroup based on 16S rRNA sequences and antigenic cross-reactivity Anderson, B. E., J. E. Dawson, et al. (1991). Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol 29(12): 2838-42.
The primary test for diagnosing CME or HME is the indirect fluorescent antibody (IFA) test. This test uses the etiologic agents Ehrlichia canis or E. chaffeensis, respectively, to diagnose infection. The IFA test, however, has serious limitations. The IFA test is subject to false positives because the antigens are whole infected cells which comprise many nonspecific proteins that can cross-react with sera from some patients. The IFA test is also subject to false negatives because IFA antigens are unstable and may become inactivated during storage. In addition the IFA test requires a special equipment to perform the test. For example, the IFA test requires a tissue culture system for growing the bacterium that are used to prepare the antigen slides, a fluorescent microscope, and trained persons to evaluate the serum reactivity to the bacterial antigen on the slide.
Serodiagnosis is another method which has been developed to diagnose canine or human ehrlichiosis. The method involves testing the blood of the animal for antibodies immunoreactive with outer membrane proteins of these pathogens. Serodiagnosis cannot be used until the infected subject has produced such antibodies. Accordingly, serodianosis cannot be used early during the course of infection. Moreover, serodiagnosis cannot be used for detecting an ongoing infection.
Accordingly, it is desirable to have additional methods and tools which can be used for diagnosing canine and human ehrlichiosis, particularly methods and tools which can be used to detect an ongoing infection. Methods and tools which can be used to detect E. canis and E. chaffeensis in the invertebrate vectors which transmit these pathogens to their respective vertebrate hosts are also desirable.

SUMMARY OF THE INVENTION

The present invention provides tools and methods for detecting the presence of E. canis and E. chaffeensis in a sample obtained from an animal, particularly from a member of the Canidae family. The method for detecting E. canis comprises providing a p30 primer set comprising a first primer having a sequence which is complementary to a sequence on the E. canis p30 gene sense strand and a second primer which is complementary to a sequence which is complementary to a sequence on the E. canis p30 gene of antisense strand, amplifying DNA in the sample using a polymerase chain reaction and the p30 primer set, and determining the length or sequence of the PCR products, wherein the presence of a PCR product having a sequence or length which corresponds to the sequence or length of the region of the p30 gene which is located between the nucleotide sequences to which the first p30 primer and the second p30 primer bind is indicative of the presence of E. canis in the sample.
The present invention also relates to the p30 primer sets. Each p30 primer set comprises a first p30 primer and the second p30 primer, both of which are from 15 to 35 nucleotides in length. The first p30 primer, i.e., the forward primer, comprises a sequence which is complementary to a consecutive sequence, preferably of at least 14 nucleotides in length, within the following sequence: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30 primer, i.e. the reverse primer, comprises a sequence which is complementary to the inverse complement of a consecutive sequence, preferably of at least 14 nucleotides in length, contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT , SEQ ID NO.2. Such primers are useful for detecting the presence of E. canis in members of the Canidae family. Such primers are also useful for detecting the presence of E. canis DNA in samples obtained from ticks or other invertebrate carriers which feed on the vertebrate hosts.
The method for detecting E. chaffeensis comprises providing a p28 primer set comprising a first primer comprising a sequence which is complementary to a sequence on the E. chaffeensis p28 gene sense strand and a second primer which is complementary to a sequence on the E. chaffeensis p28 gene antisense strand, amplifying DNA in the sample using a polymerase chain reaction and the p28 primer set, and determining the length or sequence of the PCR products, wherein the presence of a PCR product having a sequence or length which corresponds to the sequence or length of that portion of the p28 gene which is located between the sequence to which the first p28 primer and second p28 primer bind is indicative of the presence of E. chaffeensis DNA in the sample.
The present invention also relates to the primers in the p28 primer set. The first p28 and second p28 primers are from 15 to 35 nucleotides in length. The first p28 primer, i.e. the forward primer, comprises a sequence which is substantially identical to the complement of a consecutive sequence, preferably of at least 14 nucleotides in length, within the following sequence: A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO. 3.
The second primer, i.e. the reverse primer, comprises a sequence which is complementary to the inverse complement of a consecutive sequence, preferably of at least 14 nucleotides in length, within one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO.4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO.5. Such primers are useful for detecting the presence of E. chaffeensis in samples obtained from vertebrate animals such as humans or dogs, or from the invertebrate vectors which transmit this pathogen from one vertebrate animal to another.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence of the p30 gene of the Jake strain of E. canis. (Genbank Accession No. AF082744.)
FIG. 2 is a consensus open reading frame (ORF) sequence for the E. chaffeensis p28 gene. The consensus sequence was derived from the p28 genes of the Sapulpa, Jacksonville and Arkansas isolates.
FIG. 3 shows the results of a PCR assay of peripheral blood from dogs during acute and chronic experimental CME. Three dogs were tested with the nested p30-based PCR described in the text. Buffy coat DNA preparations were assayed from two dogs prior to (lanes 1 and 3) and 21 d after (Lanes 3 and 4) inoculation with E. canis Ebony isolate-infected blood from an experimentally infected dog. The third dog (lane 5) was from the experimentally infected donor blood. The molecular size standard (M) is a 100 bp ladder (Life Technologies).
FIG. 4. Confirmation of the E. canis nested target amplicon sequence. Multiple sequence alignment of the amplicon derived from nested p30-based PCR of DNA from E. canis (Ebony isolate), the E. canis p30 target DNA sequence (Jake Isolate) and primer set 1 (ECA30-384S and ECA30-583A). The DNA sequence for ECA30-583A is the reverse complement of that oligonucleotide. The sequences were aligned and compared with the Pileup (GCG, version 10) and Boxshade programs. White letters surrounded by a black box represent bases that are identical in each sequence of the alignment.
FIG. 5. Sensitivity of the p30-based nested PCR assay. The 16S rDNA (panel A) and p30 (panel B) PCR assays were compared to determine relative sensitivities. Buffy coat DNA was isolated from an E. canis carrier dog and subjected to a dilution series for simultaneous amplification with the two assays. Lanes 1-5, respectively, are assays of the template dilutions of 1×10⁰, 10⁻¹, 10⁻², 10⁻³and 10⁻⁴both panels.
FIG. 6. Specificity of the p30-based nested PCR assay. Lane 1 is the template-free control, lanes 2-3 represent amplification of 50 ng of E. canis Ebony and Oklahoma isolates, respectively, and lanes 4-6 represent assay of 50 ng of DNA isolated from E. chaffeensis, HGE agent and E. muris, respectively. The 100 bp ladder (M) serves as the molecular size standard.
FIG. 7. Preparation of blood samples for the p30-based nested PCR assay. Blood was collected with heparin from an E. canis-infected carrier dog, divided into 0.5 ml aliquots and subjected to different preparation procedures performed in duplicate as described in the text. Samples assayed with the nested p30-based PCR included buffy coats treated with DNAzol (Lanes 1 and 2), buffy coats treated with DNAzol BD (Lanes 3 and 4), whole blood treated with DNAzol BD (Lanes 5 and 6), buffy coats subjected to proteinase K digestion followed by phenol extraction and ethanol precipitation (Lanes 7 and 8) and DNA extracted from PBMC with Qiagen spin columns (Lanes 9 and 10). A 100 bp DNA ladder (M) was used as a molecular size standard.
FIG. 8. Analysis of oligonucleotide primer sets derived from the E. canis p30 gene sequence. In the primer site plot (A), the horizontal lines represent the portion of the p30 open reading frame analyzed for optimal primer pairs. Each horizontal line represents the p30 ORF; vertical bars above the line represent the location of forward primers and vertical bars below each line represent reverse primers. The truncated multiple sequence (B) demonstrates similarity between the sequences of the forward primers and the E. chaffeensis p28 sequence clusters I, II and III to the E. canis p30 sequence. Letters surrounded by a black box represent bases identical to the E. canis sequence. Gray letters surrounded by a black box represent bases identical to E. canis and all three E. chaffeensis gene clusters. The same analysis was performed for reverse primers. The percent identical bases between the different primers and the E. chaffeensis cluster sequences were determined and are displayed in Table 1.
FIG. 9. Analysis of oligonucleotide primer sets derived from the E. chaffeensis p28 consensus sequence. The primer site plot (A) represents the location of forward and reverse primers within p28; vertical bars above the line represent the location of forward primers and vertical bars below each line represent reverse primers. The truncated multiple sequence alignments demonstrate identity between the sequences of the forward (B) and reverse (C) primers. Gray letters surrounded by a black box represent bases identical to E. canis and all three E. chaffeensis gene clusters. The percent identical bases between the different primers and the E. canis sequences were determined and are displayed in Table 5.
FIG. 10. Detection of E. chaffeensis (Arkansas isolate) with the p28-based PCR assay. Panel A demonstrates amplicons from duplicate non-optimized PCR in the presence of 50 ng of DNA from E. chaffeensis-infected DH82 cells and various primer sets that are specific to the Arkansas isolate (11_A, 13_Aand 15_A) or universal to all three p28 clusters (3_Uand 1_U), which are described in Table X. Panel B consists of male ticks of four species known to parasitize dogs that were intrastadially exposed to E. chaffeensis by allowing them to feed on on a rickettsemic dog followed by holding for 10 days in a humidity chamber. Both 1° and 2° optimized PCR assays are shown for A. americanum. PCR-positive ticks are indicated by a white “+” at the top of each lane. Note that E. chaffeensis infection was detected in 20-40% of each tick species tested. A 100 bp ladder served as the molecular size standard (M).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect,the present invention provides methods and tools for detecting the presence of Ehrlichia canis in samples obtained from a vertebrate or invertebrate animal. The method comprises amplifying the DNA contained within the sample using a primer set comprising primers which comprise sequences that are complementary to select regions of the p30 gene of E. canis and a polymerase chain reaction (PCR) to provide a pool of PCR products, and then assaying the pool for the presence or absence of a PCR product whose length or sequence indicates that PCR product corresponds to the region of the p30 gene that is flanked by the nucleotide sequences which are complementary to the first and second members of the p30 primer set. The tools are the members of the p30 primer sets. The E. canis p 30 gene belongs to the omp-1 multiple gene family and has the sequence which is set forth in Genbank as Accession No. AF082744.1 (Jake isolate), Accession No. AF082750.1 (Florida isolate), Accession No. AF082749.1(Fuzzy isolate), Accession No. AF082748.1m (DJ isolate), Accession No. AF082747.1 (Arkansas isolate), Accession No. AF082746.1 (Oklahoma isolate) and Accession No. AF082745.1 (Louisiana isolate of E. canis.) The E. canis p30 gene encodes the immunodominants ehrlichial outer membrane protein P30.
In another aspect, the present invention provides methods and tools for detecting the presence of Ehrlichia chaffeensis in samples obtained from vertebrate or invertebrate animals. The method comprises amplifying the DNA contained within the sample using a p28 primer set whose members comprise a sequence which is complementary to select regions of the p28 gene of E. chaffeensis and a polymerase chain reaction (PCR) to provide a pool of PCR products, and then assaying the pool for the presence of a PCR product whose length or sequence indicates that PCR product corresponds to the region of the p28 gene that comprises and is flanked by the nucleotide sequences to which the members of the p28 primer set bind. The tools are the members of the p28 primer sets. The E. chaffeensis p28 gene belongs to the omp-1 multiple gene family and has the sequence which is set forth in Genbank as (Accession No. AF077735.1 (St. Vincent isolate), Accession No. AF077734.1 (Sapulpa isolate), Accession No. AF077733.1 (Jax isolate), Accession No. AF077732.1 (91HE17 isolate) and Accession No. AF068234 (Arkansas isolate). The E. chaffeensis p28 gene encodes the immunodominants ehrlichial outer membrane protein P28.
Primers
The primers in the p30 primer set are based upon select sequences in the p30 gene of E. canis. The p30 gene encodes the major outer membrane protein of E. canis. The sequences of the first and second primers in the p30 primer set are distinct from sequences found in the closely related p2⁸gene in E. chaffeensis. The first primer in the p30 primer set is an oligonucleotide of from 15 to 35 nucleotides in length, preferably from 18 to 30 nucleotides in length. The second p30 primer in the E. canis primer set is an oligonucleotide of from 15 to 35 nucleotides, preferably from 18 to 30 nucleotides in length. The first p30 primer, i.e., the forward primer, comprises a sequence which is substantially identical to the complement of consecutive sequence located between nucleotides 278 and 412 of the sense strand of the open reading frame sequence of the p30 gene of E. canis. The sequence of the sense strand in this region is as follows: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTT TTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTT ACTGTTCCAA AC. SEQ ID NO.1 As used herein the term “substantially identical” means that the sequence is at least 90% identical, preferably at least 95% identical, more preferably 100% identical to a particular reference sequence, e.g., to the complement of a consecutive sequence contained within SEQ ID NO. 1. The second p30 primer, i.e., the reverse primer, comprises a sequence which is substantially identical to and the inverse of a consecutive sequence located between nucleotides 465 through 597 of the sense strand of the p30 gene of E. canis. The sequence of the second p30 primer is substantially identical to the complement of the inverse complement of a consecutive sequence contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2.
In specific embodiments, the first and second primers in the p30 primer set comprise the sequences shown in Table 1 below. The first and second primers may also comprise sequences which are shorter by one or two oligonucleotides than the sequences shown in Table 1 below. The first and second primers of the E. canis primer set may also comprise a sequence which is longer than the sequences shown in Table 1 below. Such sequences have one or two additional nucleotides attached to the 5′ end of the above-listed sequences. The additional nucleotides are selected from the group consisting of adenylic acid, guanylic acid, and combinations thereof

The sequence of the thirteen E. canis p30 primer sets shown in Table 1 below are based upon a comparison of the open-reading frame sequences of the seven E. canis and five E. chaffeensis isolates. It is expected that such primer sets will specifically amplify the target sequence of multiple E. canis isolates, but not the E. chaffeensis isolates. It is expected that the primers shown in Table 1 below are both species-universal and species-specific for E. canis. In accordance with the present invention, it has been determined that amplification of DNA isolated from E. chaffeensis, E. muris, and the HGE agent with these primer sets failed to produce a PCR product that is diagnostic of E. canis.

TABLE 1


Oligonucleotide sequences of E.
canis p30 primer sets.

Set	Forward	Reverse

1	ECA30-384S	ECA30-583A
	(ATAAACACGCTGACTTTACTGTTCC)	(GTGATGAGATAGAGCGCA
		GTACC)

2	ECA30-387S	ECA30-578A
	(AACACGCTGACTTTACTGTTCC)	(ATGGCTGCCGATGTGTGA
		TG)

3	ECA30-390S	ECA30-597A
	(ACGCTGACTTTACTGTTCCAAAC)	(ATGGCTGCCGATGTGTGA
		TG)

4	ECA30-351S	ECA30-591A
	(AACATGATTGGGATGGAAGTC)	(GCCGATGTGTGATGAGAT
		AG)

5	ECA30-350S	ECA30-591bA
	(AAACATGATTGGGATGGAAGTC)	(GCCGATGTGTGATGAGAT
		AGAG)

6	ECA30-356S	ECA30-591A
	(GATTGGGATGGAAGTCCAATAC)

7	ECA30-356S	ECA30-591bA

8	ECA30-388S	ECA30-597bA
	(ACACGCTGACTTTACTGTTCCAAAC)	(ATGGCTGCCGATGTGTGA
		TGAG)

9	ECA30-282S	ECA30-486A
	(GTGTCTCACATTTTGGTAGCTTCTC)	(CTTGGGCCACCCATTGAG
		TAAC)

10	ECA30-356S	ECA30-589A
		(CGATGTGTGATGAGATAG
		AGC)

11	ECA30-355S	ECA30-589Ba
	(TGATTGGGATGGAAGTCCAATAC)	(CGATGTGTGATGAGATAG
		AGCG)

12	ECA30-353S	ECA30-597A
	(CATGATTGGGATGGAAGTCCAATAC)	(ATGGCTGCCGATGTGTGA
		TG)

13	ECA30-278S	ECA30-484A
	(CCAAGTGTCTCACATTTTGGTAGC)	(TGGGCCACCCATTGAGTA
		AC)

The predicted annealing scores and specificity values, as compared to the p28 gene of E. chaffeensis, of these primer sets are shown in Table 2 below.

TABLE 2


Ranks of E. canis p30 primer sets with predicted annealing and specificity values.

Identity Scores to E. chaffeensis ^a

Total

Primer

Cluster I

Cluster II

Cluster III

Annealing

Set	Forward	Reverse	Forward	Reverse	Forward	Reverse	Mean^b	Score^c	Rank^d

1	0.64	0.74	0.48	0.87	0.56	0.78	0.68	52	2
2	0.59	0.45	0.55	0.65	0.55	0.60	0.56	57	1
3	0.74	0.45	0.61	0.65	0.70	0.60	0.62	58	3
4	0.81	0.55	0.86	0.75	0.71	0.70	0.73	63	5
5	0.82	0.55	0.82	0.77	0.73	0.73	0.74	64	6
6	0.91	0.55	0.86	0.75	0.77	0.70	0.76	64	7
7	0.91	0.55	0.86	0.77	0.77	0.73	0.76	65	9
8	0.68	0.45	0.60	0.68	0.68	0.64	0.62	66	4
9	0.64	0.86	0.64	0.95	0.72	0.91	0.79	67	12
10	0.91	0.62	0.86	0.81	0.77	0.71	0.78	67	10
11	0.87	0.64	0.83	0.82	0.74	0.73	0.77	68	11
12	0.88	0.45	0.84	0.65	0.80	0.70	0.72	68	8
13	0.67	0.85	0.67	0.95	0.71	0.90	0.79	68	13

^aIdentity Score = No. oligonucleotide bases identical to aligned template ÷ total No. oligonucleotide bases.
^bMean identity score for both primers of each set to all three E. chaffeensis clusters.
^cDetermined by GCG (version 10) Prime program. Sum of annealing scores for primer secondary structure, non-specific primer binding to the template sequence and self-complementarity of individual primers and complementarity between primer pairs; the lowest scores indicate the primer sets with least complementarity to sequences other than the target binding site.
^dRank determined by product of annealing score and mean identity score; lower values have higher rank.

The primers in the E. chaffeensis p28 primer set are based upon select sequences in the p28 gene of E. chaffeensis. The p28 gene encodes a major outer membrane protein of E. chaffeensis. The sequences of the first and second primers in the p28 primer set are distinct from sequences found in the closely related p30 gene in E. canis. The first primer in the p28 primer set is an oligonucleotide of from 15 to 35 nucleotides in length., preferably from 18 to 30 nucleotides in length. The second primer in the E. chaffeensis p 28 primer set is an oligonucleotide of from 15 to 35 nucleotides, preferably from 18 to 30 nucleotides in length. The first p28 primer, i.e., the forward primer, comprises a sequence which is substantially identical to the complement of consecutive sequence located between nucleotides 15 and 101 of the sense strand of the open reading frame sequence of the p28 gene of E. chaffeensis. The sequence of the sense strand in this region is as follows: A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO. 3.
The second p28 primer, i.e., the reverse primer, comprises a sequence which is substantially identical to and the inverse of a consecutive sequence located between nucleotides 341 through 365 or nucleotides 641 through 674 of the sense strand of the p28 gene of E. chaffeensis. The sequence of the second p28 primer is substantially identical to the complement of the inverse complement of a consecutive sequence contained within one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO. 4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO. 5.

In specific embodiments, the first and second primers in the E. chaffeensis p28 primer set comprise the sequences shown in Table 3 below. The first and second primers may also comprise sequences which are shorter by one or two oligonucleotides than the sequences shown in Table 3 below. The first and second primers of the E. chaffeensis p28 primer set may also comprise a sequence which is longer than the sequences shown in Table 3 below. Such sequences have one or two additional nucleotides attached to the 5′ end of the above-listed sequences. The additional nucleotides are selected from the group consisting of adenylic acid, guanylic acid, and combinations thereof.

TABLE 3


product: 1
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	84 AGGTAGTGGTATTAACGG 101
reverse primer (20-mer):	360 AGATACTTCAAGCTCTATTC 341

	forward reverse
primer % GC:	44.4 35.0
primer Tm (degrees Celsius):	44.4 43.6

PRODUCT

product length:	277
product % GC: 31.0
product Tm:	70.2	degrees Celsius
difference in primer Tm:	0.8	degrees Celsius
annealing score:	57
optimal annealing temperature:	47.3	degrees Celsius

Product: 2
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	84 AGGTAGTGGTATTAACGG 101
reverse primer (20-mer):	365 TCATAAGATACTTCAAGCTC 346

	forward reverse
primer % GC:	44.4 35.0
primer Tm (degrees Celsius):	44.4 44.0

PRODUCT

product length:	282
product % GC:	30.9
product Tm:	70.2	degrees Celsius
difference in primer Tm:	0.5	degrees Celsius
annealing score:	57
optimal annealing temperature:	47.4	degrees Celsius

Product: 3
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (19-mer):	50 CTTCTCTACCTGGAGTATC 68
reverse primer (21-mer):	669 GCTTATAGAGTAGCTTAAACC 649

	forward reverse
primer % GC:	47.4 38.1
primer Tm (degrees Celsius):	44.8 45.5

PRODUCT

product length:	620
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	0.8	degrees Celsius
annealing score:	61
optimal annealing temperature:	48.3	degrees Celsius

Product: 4
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	83 CAGGTAGTGGTATTAACG 100
reverse primer (19-mer):	364 CATAAGATACTTCAAGCTC 346

	forward reverse
primer % GC:	44.4 36.8
primer Tm (degrees Celsius):	43.6 42.2

PRODUCT

product length:	282
product % GC:	31.2
product Tm:	70.3	degrees Celsius
difference in primer Tm:	1.4	degrees Celsius
annealing score:	63
optimal annealing temperature:	47.0	degrees Celsius

Product: 5
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	83 CAGGTAGTGGTATTAACG 100
reverse primer (19-mer):	359 GATACTTCAAGCTCTATTC 341

	forward reverse
primer % GC:	44.4 36.8
primer Tm (degrees Celsius):	43.6 41.9

PRODUCT

product length:	277
product % GC:	31.4
product Tm:	70.3	degrees Celsius
difference in primer Tm:	1.7	degrees Celsius
annealing score:	63
optimal annealing temperature:	46.9	degrees Celsius

Product: 6
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (19-mer):	50 CTTCTCTACCTGGAGTATC 68
reverse primer (20-mer):	669 GCTTATAGAGTAGCTTAAAC 650

	forward reverse
primer % GC:	47.4 35.0
primer Tm (degrees Celsius):	44.8 42.8

PRODUCT

product length:	620
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	2.0	degrees Celsius
annealing score:	63
optimal annealing temperature:	47.7	degrees Celsius

Product: 7

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (20-mer):	55 CTACCTGGAGTATCATTTTC 74
reverse primer (21-mer):	670 GGCTTATAGAGTAGCTTAAAC 650

	forward reverse
primer % GC:	40.0 38.1
primer Tm (degrees Celsius):	44.7 45.5

PRODUCT

product length:	616
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	0.9	degrees Celsius
annealing score:	64
optimal annealing temperature:	48.3	degrees Celsius

Product: 8
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	45 AATATCTTCTCTACCTGG 62
reverse primer (19-mer):	359 GATACTTCAAGCTCTATTC 341

	forward reverse
primer % GC:	38.9 36.8
primer Tm (degrees Celsius):	40.8 41.9

PRODUCT

product length:	315
product % GC:	32.1
product Tm:	70.9	degrees Celsius
difference in primer Tm:	1.1	degrees Celsius
annealing score:	65
optimal annealing temperature:	47.0	degrees Celsius

Product: 9
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	15 AGTTTTCATAACAAGTGC 32
reverse primer (19-mer):	364 CATAAGATACTTCAAGCTC 346

	forward reverse
primer % GC:	33.3 36.8
primer Tm (degrees Celsius):	42.5 42.2

PRODUCT

product length:	350
product % GC:	31.4
product Tm:	70.9	degrees Celsius
difference in primer Tm:	0.3	degrees Celsius
annealing score:	66
optimal annealing temperature:	47.4	degrees Celsius

Product: 10
[DNA] 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	15 AGTTTTCATAACAAGTGC 32
reverse primer (19-mer):	359 GATACTTCAAGCTCTATTC 341

	forward reverse
primer % GC:	33.3 36.8
primer Tm (degrees Celsius):	42.5 41.9

PRODUCT

product length:	345
product % GC:	31.6
product Tm:	70.9	degrees Celsius
difference in primer Tm:	0.6	degrees Celsius
annealing score:	66
optimal annealing temperature:	47.3	degrees Celsius

Product: 11
[DNA] = 50.000 nM [salt] 50.000 mM

PRIMERS

	5′ 3′
forward primer (25-mer):	50 CTTCTCTACCTGGAGTATCATTTTC 74
reverse primer (26-mer):	662 GAGTAGCTTAAACCTAACTTTCCTTG 637

	forward reverse
primer % GC:	40.0 38.5
primer Tm (degrees Celsius):	50.9 52.1

PRODUCT

product length:	613
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	1.2	degrees Celsius
annealing score:	67
optimal annealing temperature:	50.1	degrees Celsius

Product: 12
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (19-mer):	50 CTTCTCTACCTGGAGTATC 68
reverse primer (22-mer):	662 GAGTAGCTTAAACCTAACTTTC 641

	forward reverse
primer % GC:	47.4 36.4
primer Tm (degrees Celsius):	44.8 46.6

PRODUCT

product length:	613
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	1.8	degrees Celsius
annealing score:	67
optimal annealing temperature:	48.3	degrees Celsius

Product: 13
[DNA] = 50.000 nM [salt] 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	45 AATATCTTCTCTACCTGG 62
reverse primer (18-mer):	365 TCATAAGATACTTCAAGC 348

	forward reverse
primer % GC:	38.9 33.3
primer Tm (degrees Celsius):	40.8 40.2

PRODUCT

product length:	321
product % GC:	31.8
product Tm:	70.8	degrees Celsius
difference in primer Tm:	0.6	degrees Celsius
annealing score:	68
optimal annealing temperature:	46.7	degrees Celsius

Product: 14
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	45 AATATCTTCTCTACCTGG 62
reverse primer (19-mer):	364 CATAAGATACTTCAAGCTC 346

	forward reverse
primer % GC:	38.9 36.8
primer Tm (degrees Celsius):	40.8 42.2

PRODUCT

product length:	320
product % GC:	31.9
product Tm:	70.9	degrees Celsius
difference in primer Tm:	1.4	degrees Celsius
annealing score:	69
optimal annealing temperature:	47.0	degrees Celsius

Product: 15
[DNA] = 50.000 nM [salt] 50.000 mM

PRIMERS

	5′ 3′
forward primer (24-mer):	51 AATATCTTCTCTACCTGG 62
reverse primer (19-mer):	364 CATAAGATACTTCAAGCTC 645

	forward reverse
primer % GC:	37.5 38.5
primer Tm (degrees Celsius):	49.8 51.3

PRODUCT

product length:	620
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	1.5	degrees Celsius
annealing score:	69
optimal annealing temperature:	49.8	degrees Celsius

Product: 16
[DNA[ = 50.000 nM [salt] =50.000 mM

PRIMERS

	5′ 3′
forward primer (22-mer):	53 CTCTACCTGGAGTATCATTTTC 74
reverse primer (22-mer):	670 GGCTTATAGAGTAGCTTAAACC 649

	forward reverse
primer % GC:	40.9 40.9
primer Tm (degrees Celsius):	47.7 48.1

PRODUCT

product length:	618
product % GC:	30.1
product Tm:	71.2	degrees Celsius
difference in primer Tm:	0.4	degrees Celsius
annealing score:	71
optimal annealing temperature:	49.2	degrees Celsius

Product: 17
[DNA] = 50.000 nM [salt] 50.000 mM

PRIMERS

	5′ 3′
forward primer (22-mer):	53 CTCTACCTGGAGTATCATTTTC 74
reverse primer (22-mer):	662 GAGTAGCTTAAACCTAACTTTC 641

	forward reverse
primer % GC:	40.9 36.4
primer Tm (degrees Celsius):	47.7 46.6

PRODUCT

product length:	610
product % GC:	30.0
product Tm:	71.1	degrees Celsius
difference in primer Tm:	1.1	degrees Celsius
annealing score:	71
optimal annealing temperature:	48.8	degrees Celsius

Product: 18
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (18-mer):	57 ACCTGGAGTATCATTTTC 74
reverse primer (18-mer):	674 TCTGGGCTTATAGAGTAG 657

	forward reverse
primer % GC:	38.9 44.4
primer Tm (degrees Celsius):	42.6 43.0

PRODUCT

product length:	618
product % GC:	30.1
product Tm:	71.2	degrees Celsius
difference in primer Tm:	0.4	degrees Celsius
annealing score:	73
optimal annealing temperature:	47.7	degrees Celsius

Product: 19
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (19-mer):	50 CTTCTCTACCTGGAGTATC 68
reverse primer (18-mer):	673 CTGGGCTTATAGAGTAGC 656

	forward reverse
primer % GC:	47.4 50.0
primer Tm (degrees Celsius):	44.8 45.1

PRODUCT

product length:	624
product % GC:	30.3
product Tm:	71.2	degrees Celsius
difference in primer Tm:	0.4	degrees Celsius
annealing score:	73
optimal annealing temperature:	48.4	degrees Celsius

Product: 20
[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

	5′ 3′
forward primer (20-mer):	55 CTTCTCTACCTGGAGTATC 68
reverse primer (18-mer):	673 CTGGGCTTATAGAGTAGC 656

	forward reverse
primer % GC:	40.0 50.0
primer Tm (degrees Celsius):	44.7 45.1

PRODUCT

product length:	619
product % GC:	30.2
product Tm:	71.2	degrees Celsius
difference in primer Tm:	0.5	degrees Celsius
annealing score:	75
optimal annealing temperature:	48.3	degrees Celsius

Sample

For the vertebrate hosts the sample is a tissue sample or bodily fluid, such as for example, buffy coats and peripheral blood mononuclear cells. For the invertebrate vectors which may transmit the pathogen from one vertebrate host to another, the sample can be dissected from ticks (e.g., midgut, salivary glands and hemolymph), ticks can be cut into pieces, and ticks can be frozen and smashed in preparation for PCR assay. Further preparation of tick tissues may involve just heating the sample, digesting the samples with proteinase or isolating pure DNA from the tick tissues.
Methods
Optionally, DNA is extracted from the sample using standard methods. For example, DNA may be extracted from fluids using commercially available PCR filters such as, for example, the Isocode filters which are available from Schleicher and Schuell, New Hampshire. Methods of extracting DNA from tissue samples, are also described in Maniatis, T., J. Sambrook, and E. F. Fritsch. 1989. Molecular Cloning: a Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N. Y., which is specifically incorporated herein by reference. An optimal method for vertebrates involves collection of whole blood in the presence of an anticoagulant such as heparin, collecting the buffy coat fraction from the whole blood, removal of host hemoglobin by hypotonic lysis of erythrocytes remaining with the buffy coat followed by several washings of the buffy coat prior to preparation of the template for PCR assay. For detecting the pathogens in the invertebrate vectors, DNA is extracted from tick halves, salivary glands and midgut by digestion with proteinase K in the presence of nonionic detergents such as Tween-20 and NP-40 followed by heat inactivation of the proteinase and an optional procedure to extract proteins with phenol and chloroform followed by DNA precipitation by salt and ethanol.
The DNA is then amplified with the one of the primer sets at PCR conditions equivalent to or comparable to denaturation for 0.5 to 1.0 minute at 94° C. in a solution, followed by annealing at about 50-72° C. for 0.5 to 1.0 minutes. As recognized in the art, suitable stringency conditions can be attained by varying a number of factors such as the length and nature, i.e., DNA or RNA, of the primer; the length and nature of the target sequence; the concentration of the salts and other components of the PCR solution; the temperature and time of each step, e.g., denaturation, annealing, and elongation. All of these factors may be varied to generate conditions of stringency which are equivalent to the conditions listed above. Changes in stringency are accomplished primarily through the manipulation of annealing temperature and time.
Thereafter, the size of the PCR products is determined. Preferably the size of the PCR products is determined using gel electrophoresis and a plurality of DNA standards of varying sizes. The PCR products and DNA standards that are present on the gel are visualized using standard techniques, such as ethidium bromide staining. Optionally, the PCR products may be separated, preferably by gel electrophoresis and the sequence of the PCR products determined using standard techniques.
To further enhance the sensitivity and specificity of the method for detecting E. canis, a nested PCR assay which employs two different E. canis primer sets is used. In such assay, the sample is first amplified with a primer set comprising two external primers, i. e. ECA30-351S and ECA30-591A, ECA30-350S and ECA30-591bA, ECA30-356S and ECA30-591A, ECA30-356S and ECA30-591Ba, ECA30-356S and ECA30-589A, ECA30-355S and ECA30-589bA, and ECA30-353S and ECA30-597A. Thereafter, the products of the first PCR are amplified with a second primer set that comprises internal primers, eg. ECA30-384S and ECA30. A highly preferred primer set for this second amplification comprises a first primer comprising the sequence 5′-ATAAACACGCTGACTTTACTGTTCC-3′ and a second primer of comprising the sequence 5′-GTGATGAGATAGAGCGCAGTACC-3′. Following the second amplification, the size or sequence of the PCR products are determined to detect a PCR product which indicates that a DNA molecule having a sequence which corresponds to the region of the p30 gene that is located between the consecutive sequences to which the internal primers bind is present in the sample.
To further enhance the sensitivity and specificity of the method for detecting E. chaffeensis, a nested PCR assay which employs two different E. chaffeensis primer sets is used. In such assay, the sample is first amplified with a primer set that comprises external primers, i.e. E. chaffeensis primer set 3 A highly preferred primer set for this first amplification comprises a first primer comprising the sequence CTTCTCTACCTGGAGTATC and a second primer comprising the sequence GCTTATAGAGTAGCTTAAACC. Thereafter, the products of the first PCR are amplified with a second primer set that comprises two internal primers, e.g. E. chaffeensis primer set 1. A highly preferred primer set for this second amplification comprises a first primer comprising the sequence AGGTAGTGGTATTAACGG and a second primer which comprises the sequence AGATACTTCAAGCTCTATTC. Following the second amplification, the size or sequence of the PCR products are determined to detect a PCR product which indicates that a DNA molecule having a sequence which corresponds to 277 bp of the p28 gene is present in the sample.

EXAMPLES

The following examples are for purposes of illustration only and are not intended to limit the scope of the invention as defined in the claims which are appended hereto. The references cited in this document are specifically incorporated herein by reference.

Example 1

A. Obtaining DNA from Blood Samples of Infected Animals
Two colony-reared Beagle dogs that were seronegative for exposure to E. canis by IFA were inoculated with heparinized blood from a donor dog infected with E. canis (Ebony isolate). Heparinized blood was collected from each animal and split into 0.5 ml aliquots and subjected to the following treatments: (1) DNAzol extraction of buffy coat, (2) DNAzol-BD (Molecular Research Center) extraction of whole blood, (3) DNAzol-BD extraction of buffy coat, (4) spin-column purification of peripheral blood mononuclear cell DNA with a QIAamp Kit (Qiagen) and (5) phenol/chloroform extraction followed by ethanol precipitation of buffy coat samples. All sample preparation methods were done in duplicate and in two trials. Buffy coats were isolated from the heparinized whole blood after centrifugation at 1000×g for 20 min, except for those to be purified with spin-columns, which were isolated after isopycnic centrifugation as described in Iqbal, Z., and Y. Rikihisa. 1994. Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs. Vet. Microbiol. 42:281-7. All DNA isolations with commercial kits were performed in strict accordance to the manufacturer's instructions. DNAzol and DNAzol-BD isolations were performed in the presence of polyacryl carrier (Molecular Research Center) as recommended by the manufacturer.
The remaining method was a modification of a protocol that was previously described in Stich, R. Wet al. 1991. Preliminary development of a polymerase chain reaction assay for Anaplasma marginale in ticks. Biotechnol. Techniq. 5:269-274.; and Stich, R. W., et al 1993. Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction. J. Med. Entomol. 30:789-94. Briefly, the buffy coats were removed from whole blood and transferred to a 1.5 ml microfage tube. Erythrocytes remaining in the buffy coat were lysed with two volumes of TE (10 mM Tris.HCl, pH 8.0, 1 mM EDTA), and the hemoglobin was removed by washing pelleted cells (13,000×g for 1 min) three times in TE. The final cell suspension was in 400 μl of RPMI 1640 containing 100 μg/ml proteinase K and 0.45% (v/v) NP-40 and 0.45% (v/v) Tween-20, and the protein was digested for 1-2 hr at 55° C. Digests were extracted one time each with equal volumes of buffer-saturated phenol (pH>7.5) (Life Technologies), phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1). DNA was precipitated by adding 1/10 volume of 3 M sodium acetate and 2.5 volumes of absolute ethanol (−20° C.) and stored overnight at −20° C. The DNA samples were centrifuged at 13,000×g for 20 min at 4° C., allowed to air dry in a sterile, bleach-treated cabinet and resuspended in 25 μl of HPLC grade H₂O. The samples containing E. canis DNA were diluted empirically throughout the optimization process.
B. Nested PCR
The primers used in the first PCR reaction is set 12 shown in Table 1 above. The primers used in the second PCT reaction is set 1 shown in Table 1 above. PCR was performed with a Perkin-Elmer 2400 thermal cycler. Master mixes, made with the PE Biosystems Reagents (Foster City, Calif.), were divided into 50 or 25 μl final reaction volumes containing PCR Gold buffer, 0.8 mM dNTP mix and specified amounts of MgCl₂, primers, Amplitaq-Gold DNA Polymerase and 10% (v/v) template. The reaction profile, except for stated exceptions, consisted of 95° C. for 10 min followed by 35 cycles of 94° C. for 1.0 min, 60° C. for 0.5 min, and 72° C. for 0.5 min followed by a final extension at 72° C. for 7.0 min.
The following PCR parameters were progressively optimized: (1) annealing temperature (37°, 50°, 55°, 60°, 65° and 70° C. with 1.5 mM MgCl₂, 0.5 μM primers and 0.025 U/μl Amplitaq-Gold), (2) MgCl₂concentration (1.0-4.0 mM MgCl₂in 0.5 mM increments), (3) primer concentration (0.1-1.0 μM in 0.1 μM increments), (4) Amplitaq-Gold concentration (0.01-0.1 U/μl in 0.01 U/μl increments) and (5) cycle number (25-65 cycles in 5 cycle increments).
C. Analysis of PCR Products
PCR product (20 μl) was added to 5 μl of loading buffer (40% (w/v) sucrose, 89 mM Tris, 89 mM boric acid, 2 mM EDTA) and electrophoresed on a 1.5% (w/v) agarose gel with 0.5 μg/ml ethidium bromide in 1× TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 60-80 V for 1-1.5 hr. A 100 bp ladder (Life Technologies, Rockville, Md.) was used as the molecular weight standard. DNA bands were visualized with ultraviolet light and documented using an Alpha Innotech 2000 gel imaging system.

RESULTS

Amplification of the 200 bp target sequence by nested PCR of E. canis-infected peripheral blood from dogs with acute experimental CME.
A 200 bp band was amplified from DNA prepared from blood samples collected from three dogs during acute or chronic experimental CME (FIG. 1). This band was not observed from blood collected prior to infection of the two dogs assayed during acute CME. The DNA sequence of this amplicon was found to be identical to the 200 bpp3O target sequence and the reverse complement of ECAR1583A (FIG. 2).
Optimum Conditions for Isolating DNA from Blood Samples
Several approaches to blood preparation for PCR assay were compared with duplicate samples in two trials. Venous blood was collected with heparin from an experimentally infected E. canis carrier, equal aliquots that were subjected to different protocols for the preparation of template for PCR. Inhibition of PCR by hemoglobin is well known, thus removing hemoglobin from the blood sample is a major concern of any procedure used to assay these samples with PCR. Commercially available chemical and spin column kits for DNA isolation did provide some positive results with buffy coat and whole blood samples, but these treatments were inconsistent, failing to result in amplification of DNA in both duplicates of both trials. However, buffy coat isolation followed by lysing residual erythrocytes, removal of hemoglobin, protein digestion, protein extraction and DNA precipitation was the only procedure that resulted in amplification of the 200 bp target in both duplicates of both trials (FIG. 5). Moreover, this method has been used to detect E. canis in peripheral blood of a carrier dog for over one year (data not shown).
Optimization of the p30-based PCR assay for E. canis. Initial optimization experiments with primer set 2 resulted in the amplification of multiple bands, thus this primer set is less preferred. The remaining assays employed primer sets 1 and 12. For the primary reaction with set 12, optimum PCR conditions were determined to be 1.5 mM MgCl₂, 0.2 μM of each primer, 0.04 units/μl of Amplitaq-Gold and, after enzyme activation at 95° C. for 10 min, 55 cycles with 94, 65 and 72° C. denaturation, annealing and extension temperatures for 30 sec each. For the secondary or nested PCR with internal primer set 1, optimum reaction conditions were determined to be 2.5 mM MgCl₂, 0.5 μM of each primer, 0.03 units/μl of Amplitaq-Gold, 10% (vol/vol) of the appropriate primary reaction with primer set 12 and 40 cycles with 94, 60 and 72° C. denaturation, annealing and extension temperatures for 30 sec each.
To confirm that the 200 bp amplicon associated with E. canis infection originated from the target DNA sequence, several p30-based nested PCR assays of buffy coat DNA from an E. canis-infected dog were pooled and the amplicons isolated with the QIAquik PCR Purification Kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. The purified amplicon was submitted to The Ohio State University Neurobiotechnology DNA Sequencing Facility for cycle sequencing in the presence of the primer, ECAF1384S.
The optimum number of cycles for both reactions, particularly the primary reaction, were somewhat greater than expected. One explanation for this may be the enzyme used, Amplitaq-Gold, which requires activation at 95° C. for 10 min prior to the amplification cycles. It is possible that a portion of this enzyme remains inactive until the amplification cycles are underway; thus additional active enzyme becomes available in the course of the reaction, increasing the number of cycles required for optimal PCR.
Specificity and Sensitivity.
The sensitivity of this p30-based assay was compared to that of the previously reported 16S rDNA-based assay as described in Wen, B., Y. Rikihisa, J. M. Mott, R. Greene, H. Y. Kim, N. Zhi, G. C. Couto, A. Unver, and R. Bartsch. 1997. Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J. Clin. Microbiol. 35:1852-5.The buffy coat fraction was removed from 5 ml of heparinized blood from an experimentally infected dog during acute CME, and subjected to DNA isolation with DNAzol (Molecular Research Center) according to the manufacturer's instructions. A tenfold dilution series of the buffy coat DNA was then tested with both the p30- and the 16S rDNA-based PCR assays.
Specificity of the p30-based assay was tested by attempts to amplify 50 ng samples of DNA isolated from E. muris, E. chaffeensis and HGE agent in experimentally infected host cells. DNA. E. chaffeensis and E. muris are the two known species most closely related to E. canis (based on 16S rDNA sequence homology) Faint bands at sizes other than that of the target sequence were occasionally observed, indicating that similar omp-1 sequences may be amplified under these conditions, but the robust 200 bp band was only observed with E. canis template.

Example 2

E. chaffeensis p28 ORF sequences were compared to identify prospective oligonucleotide primer sequences for a PCR assay. Design of the primers for this PCR assay was similar to that described for E. canis, except that identities of these primers to the three clusters of p28 sequences among the E chaffeensis isolates were also considered by dividing the product of the annealing and E. canis identity scores by the mean of the three E. chaffeensis cluster identity scores. Primers were designed from the E. chaffeensis Arkansas isolate p28 sequence. These primer sequences were aligned with the homologous portions of the E. canis p30 geme and the three p28 clusters and the identity of these sequences determined Total annealing scores, identity to the different p28 clusters and identity to p30 were then used to rank these primer sets. A second approach to primer design involved the design of primers to the consensus sequence of all three E. chaffeensis p28 clusters through less stringent annealing score parameters. These primers, which are shown in Table 3 above are expected to be universal for all five E. chaffeensis isolates that composed these three clusters.

Primers for E. chaffeensis were designed with the Wisconsin Package (GCG) Version 10 software suite (Madison, Wisc.). This was accomplished with a multiple sequence alignment of the target template from isolates representing all three clusters of E. chaffeensis of and the E. canis p30 sequence. Initially optimal primer sequences could not be obtained from the consensus of all three p28 sequences, thus primers were designed from the E. chaffeensis Arkansas isolate p28 sequence since this is the isolate to be used in the proposed investigation. Twenty-five sets of primers were then identified. These primer sequences were aligned with the homologous portions of p30 and the three p28 clusters and the identity of these sequences determined. Total annealing scores, identity to the different p28 clusters and identity to p30 were then used to rank these primer sets. Three primer sets were chosen for development of a p28-based assay by virtue of their overall rank and relative positions flanking or internal to other primer sets for nested PCR and found to amplify E. chaffeensis DNA from infected canine cells. Two of the E. chaffeensis Arkansas isolate-specific primer sets were optimized and used as a nested PCR assay to detect the pathogen in intrastadially infected male ticks of four species known to parasitize dogs.

TABLE 4


Ranks of E. chaffeensis p28 primer sets with predicted annealing and specificity values.^a

Identity Scores to E. chaffeensis

Identity Scores to

Primers

Anealing

Cluster I

Cluster II

Cluster III

E. canis

Set

Fwd.

Rev.

Score

Fwd.

Rev.

Fwd.

Rev.

Fwd.

Rev.

Mean

Fwd.

Rev.

Mean

Rank

1^b	67-85	341-320	55	1.0	1.0	0.90	0.95	1.0	1.0	0.98	0.68	0.91	0.80	8
2	58-78	321-302	58	1.0	0.95	0.95	0.95	1.0	1.0	0.98	0.57	0.90	0.74	7
3	58-78	314-294	58	1.0	0.90	0.95	0.90	1.0	1.0	0.96	0.57	0.86	0.72	5
4	61-82	339-319	58	1.0	0.90	0.91	0.95	1.0	1.0	0.96	0.64	0.86	0.75	10
5	61-82	315-294	59	1.0	0.91	0.91	0.91	1.0	1.0	0.96	0.64	0.86	0.75	15
6	61-82	320-301	59	1.0	1.0	0.91	0.95	1.0	1.0	0.98	0.64	0.90	0.77	16
7	60-80	314-294	60	1.0	0.90	0.95	0.90	1.0	1.0	0.96	0.62	0.86	0.74	13
8	59-79	314-294	60	1.0	0.90	0.95	0.90	1.0	1.0	0.96	0.62	0.86	0.74	14
9	58-79	322-302	60	1.0	0.95	0.95	0.95	1.0	1.0	0.98	0.59	0.90	0.75	11
10	58-78	320-301	60	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.57	0.90	0.74	9
*11^b	58-78	341-320	61	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.57	0.91	0.74	12
12	59-79	320-301	62	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.62	0.90	0.76	19
*13^b′	78-99	314-294	62	1.0	0.90	0.95	0.90	1.0	1.0	0.96	0.41	0.86	0.64	2
14	59-80	320-301	62	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.64	0.90	0.77	20
15	58-78	352-331	62	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.57	0.77	0.67	3
16	58-78	339-319	64	1.0	0.90	0.95	0.95	1.0	1.0	0.97	0.57	0.86	0.72	18
17	78-99	341-320	64	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.41	0.91	0.66	4
18	58-78	313-293	65	1.0	0.95	0.95	0.90	1.0	1.0	0.97	0.57	0.95	0.76	25
19	60-80	341-320	64	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.62	0.91	0.77	21
20	78-99	352-331	65	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.41	0.77	0.59	1
21	61-82	314-293	65	1.0	0.91	0.91	0.91	1.0	1.0	0.96	0.64	0.86	0.75	24
22	59-79	341-320	65	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.62	0.91	0.77	23
23	59-80	352-331	66	1.0	1.0	0.95	0.95	1.0	1.0	0.98	0.64	0.77	0.71	17
24^b′	78-99	314-293	66	1.0	0.91	0.95	0.91	1.0	1.0	0.96	0.41	0.86	0.64	6
25	60-80	339-319	66	1.0	0.90	0.95	0.95	1.0	1.0	0.97	0.62	0.86	0.74	22

^aAll values determined as described for Table 1, except for Rank, which was determined by the product of annealing score and mean E. canis identity score, divided by mean E. chaffeensis annealing score; lower values have higher rank.
^b,b′Nested primer sets.
*Primer sets chosen for optimization.

Primers derived from both approaches were chosen for use in a p28-based assay by virtue of their overall rank and relative positions flanking or internal to other primer sets for nested PCR. These candidate primer were synthesized and they were all tested with 50 ng of DNA from E. chaffeensis-infected DH82 cells and Life Technologies reagents at concentrations of 1× PCR buffer, 1.5 mM MgCl₂, 0.5 μM of each primer and 0.025 U/μl Platinum Taq DNA Polymerase at an initial incubation period at 94° C. for 2 min followed by 25 cycles of 94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 1 min 7 min final extension step at 72° C. Each primer set tested amplified a clean, robust amplicon of the expected size under unoptimized conditions. Primer sets F58/R341 and F78/R314 were chosen first for optimization due to the more stringent parameters used for their selection.
Optimum parameters for primer sets F58/R341 and F78/R314 were then used to assay canine and tick host samples. Dog number 146 was inoculated iv. with DH82 cells that were infected in vitro with E. chaffeensis (Arkansas isolate). Once the dog was PCR-positive by the 16S rDNA-based PCR assay and seroconverted, it was infested with 150 adult males of A. americanum, and 10 each of A. maculatum, D. variabilis and R. sanguineus. These ticks were allowed to simultaneously acquisition feed seven days prior to their removal and incubation in a humidity chamber for another 10 days. The held ticks were then placed at 37° C. at 100% rh for 80 hr before they were aseptically bisected and digested with proteinase K in the presence of nonionic detergents. These ticks (2.5 μl) were then assayed with E. chaffeensis p28-specific nested primer sets 11_A(F58/R341)and 13_A(F78/R314).
Samples from each tick species fed tested PCR-positive at a frequency of 20-40% with the nested assay. These results are the first to our knowledge to demonstrate detection of E. chaffeensis in experimentally infected ticks, only the second time to detect E. chaffeensis in individual ticks and the first detection of this pathogen in A. maculatum or R. sanguineus.

Further sequence analysis with less stringent criteria did reveal 20 primer sequences complementary to the p28 consensus sequence of all three clusters. Two sets of the consensus primers (See * in Table 5 below) were also tested and found to amplify robust amplicons from E. chaffeensis DNA.

TABLE 5


Ranks of universal E. chaffeensis p28
primer sets with predicted values.

			Identity Scores to
	Primers	Anealing	E. canis

Set	Fwd.	Rev.	Score	Fwd.	Rev.	Mean	Rank

*1	84-101	360-341	57	0.5	0.8	0.65	1
2	84-101	365-346	57	0.5	0.8	0.65	1
*3	50-68	669-649	61	0.58	0.67	0.62	3
4	83-100	364-346	63	0.44	0.79	0.62	5
5	83-100	359-341	63	0.44	0.79	0.62	5
6	50-68	669-650	63	0.58	0.65	0.61	4
7	55-74	670-650	64	0.65	0.67	0.66	7
8	45-62	359-341	65	0.61	0.79	0.70	10
9	15-32	364-346	66	0.78	0.79	0.78	19
10	15-32	359-341	66	0.78	0.79	0.78	19
11	50-74	662-637	67	0.64	0.73	0.69	11
12	50-68	662-641	67	0.58	0.68	0.63	8
13	45-62	365-348	68	0.61	0.78	0.69	13
14	45-62	364-346	69	0.61	0.79	0.70	15
15	51-74	670-645	69	0.67	0.58	0.62	9
16	53-74	670-649	71	0.69	0.64	0.66	12
17	53-74	662-641	71	0.67	0.72	0.69	17
18	57-74	674-657	73	0.67	0.72	0.69	14
19	50-68	673-656	73	0.58	0.72	0.65	14
20	55-74	673-656	75	0.65	0.72	0.69	18

Claims

1. A method for detecting E. canis in a sample obtained from an animal, comprising

(a) providing a primer set comprising:

(i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:

SEQ ID NO.1 CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC., and

(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within the following sequence:

SEQ ID NO.2 GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT,.;

(b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and

(c) determining the length or sequence of the PCR products of step (b), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. canis p30 gene which is located between the regions to which the forward primer and the reverse primer bind is indicative of the presence of E. canis in the sample.

2. The method of claim 1 wherein the consecutive sequence is at least 14 nucleotides in length.

3. The method of claim 1 wherein the forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:

PAIR 1: ATAAACACGCTGACTTTACTGTTCC, S, SEQ ID NO. 6 GTGATGAGATAGAGCGCAGTACC,.; SEQ ID NO. 7 PAIR 2 AACACGCTGACTTTACTGTTCC,, SEQ ID NO. 8 ATGGCTGCCGATGTGTGATG,, SEQ ID NO. 9 PAIR 3: ACGCTGACTTTACTGTTCCAAAC,, SEQ ID NO. 10 ATGGCTGCCGATGTGTGATG,, SEQ ID NO. 11 PAIR 4: AACATGATTGGGATGGAAGTC,, SEQ ID NO. 12 GCCGATGTGTGATGAGATAG,; SEQ ID NO. 13 PAIR 5: AAACATGATTGGGATGGAAGTC,, SEQ. ID NO. 12 GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 6: GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 GCCGATGTGTGATGAGATAG,; SEQ ID NO. 11 PAIR 7: GATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 13 GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 8: ACACGCTGACTTTACTGTTCCAAAC,; SEQ ID NO. 15 ATGGCTGCCGATGTGTGATGAG,; SEQ ID NO. 16 PAIR 9: GTGTCTCACATTTTGGTAGCTTCTC,, SEQ ID NO. 17 CTTGGGCCACCCATTGAGTAAC,; SEQ ID NO. 18 PAIR 10 GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 CGATGTGTGATGAGATAGAGC,; SEQ ID NO. 19 PAIR 11: TGATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 20 CGATGTGTGATGAGATAGAGCG,, SEQ ID NO. 21 PAIR 12: CATGATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 21 ATGGCTGCCGATGTGTGATG,; SEQ ID NO. 11 PAIR 13: CCAAGTGTCTCACATTTTGGTAGC,, SEQ ID NO. 22 TGGGCCACCCATTGAGTAAC,. SEQ ID NO. 23

4. The method of claim 3 wherein the primer set is pair 12.

5. The method of claim 3 wherein the primer set is pair 1.

6. A method for detecting E. canis in a sample obtained from an animal, comprising

(a) providing a first primer set comprising:

(b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction to provide a pool of PCR products,

(c) amplifying the products of step (b) using a polymerase chain reaction and a second primer set comprising:

wherein one or both of the primers of the second primer set are internal to the primers of the first primer set; and

(d) determining the length or sequence of the PCR products of step (c), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. canis p30 gene which is located between the regions to which the forward primer of the second primer set and the reverse primer of the second primer set bind is indicative of the presence of E. canis in the sample.

7. A method for detecting E. chaffeensis in a sample obtained from an animal, comprising

(a) providing a primer set comprising:

SEQ ID NO.3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and

(ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within one of the following two sequences:

SEQ ID NO.4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,

(c) determining the length or sequence of the PCR products of step (b), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. chaffeensis p28 gene which is located between the regions to which the forward primer and the reverse primer bind is indicative of the presence of E. chaffeensis in the sample.

8. The method of claim 7 wherein the consecutive sequence is at least 14 nucleotides in length.

9. The method of claim 7 wherein the forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:

10. The method of claim 9 wherein the primer set is pair 1.

11. The method of claim 9 wherein the primer set is pair 3.

12. A method for detecting E. chaffeensis in a sample obtained from an animal, comprising

(a) providing a first primer set comprising:

providing a primer set comprising:

SEQ ID NO. 3 A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and

(d) determining the length or sequence of the PCR products of step (c), wherein the presence of a PCR product having a length or sequence which corresponds to the length or sequence, respectively, of that region of the E. chaffeensis p28gene which is located between the regions to which the forward primer of the second primer set and the reverse primer of the second primer set bind is indicative of the presence of E. chaffeensis in the sample.

13. A primer set for detecting E. canis in a sample, said primer set comprising:

(a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:

14. The primer set of claim 13 wherein forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:

PAIR 1: ATAAACACGCTGACTTTACTGTTCC, S, SEQ ID NO. 6 GTGATGAGATAGAGCGCAGTACC,.; SEQ ID NO. 7 PAIR 2 AACACGCTGACTTTACTGTTCC,, SEQ ID NO. 8 ATGGCTGCCGATGTGTGATG,, SEQ ID NO. 9 PAIR 3: ACGCTGACTTTACTGTTCCAAAC,, SEQ ID NO. 10 ATGGCTGCCGATGTGTGATG,, SEQ ID NO. 11 PAIR 4: AACATGATTGGGATGGAAGTC,, SEQ ID NO. 12 GCCGATGTGTGATGAGATAG,, SEQ ID NO. 13 PAIR 5: AAACATGATTGGGATGGAAGTC,, SEQ. ID NO. 12 GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 6: GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 GCCGATGTGTGATGAGATAG,; SEQ ID NO. 11 PAIR 7: GATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 13 GCCGATGTGTGATGAGATAGAG,; SEQ ID NO. 14 PAIR 8: ACACGCTGACTTTACTGTTCCAAAC,; SEQ ID NO. 15 ATGGCTGCCGATGTGTGATGAG,; SEQ ID NO. 16 PAIR 9: GTGTCTCACATTTTGGTAGCTTCTC,, SEQ ID NO. 17 CTTGGGCCACCCATTGAGTAAC,; SEQ ID NO. 18 PAIR 10 GATTGGGATGGAAGTCCAATAC,, SEQ. ID. NO. 13 CGATGTGTGATGAGATAGAGC,; SEQ ID NO. 1; PAIR 11: TGATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 20 CGATGTGTGATGAGATAGAGCG,, SEQ ID NO. 21 PAIR 12: CATGATTGGGATGGAAGTCCAATAC,, SEQ ID NO. 21 ATGGCTGCCGATGTGTGATG,; SEQ ID NO. 11 PAIR 13: CCAAGTGTCTCACATTTTGGTAGC,, SEQ ID NO. 22 TGGGCCACCCATTGAGTAAC,. SEQ ID NO. 23

15. The primer set of claim 14 wherein the primer set comprises pair 1.

16. The primer set of claim 14 wherein the primer set comprises pair 12.

17. A primer set for detecting E. chaffeensis in a sample, said primer set comprising a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is the complement of a consecutive sequence within the following sequence:

SEQ ID NO. 3 (a) A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG,; and +P1 (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a consecutive sequence within one of the following two sequences: SEQ ID NO. 4 CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA,, or SEQ ID NO. 5 C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA,.

18. The primer set of claim 17 wherein the forward primer and the reverse primer, respectively, comprise one of the following pairs of sequences:

PAIR 1 AGGTAGTGGTATTAACGG, SEQ ID NO 24 AGATACTTCAAGCTCTATTC,; SEQ ID NO. 25 PAIR 2: AGGTAGTGGTATTAACGG,, SEQ ID NO TCATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 3 CTTCTCTACCTGGAGTATC,, SEQ ID NO GCTTATAGAGTAGCTTAAACC,; SEQ ID NO PAIR 4: CAGGTAGTGGTATTAACG,, SEQ ID NO CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 5 CAGGTAGTGGTATTAACG,, SEQ ID NO GATACTTCAAGCTCTATTC,; SEQ ID NO PAIR 6 CTTCTCTACCTGGAGTATC,, SEQ ID NO GCTTATAGAGTAGCTTAAAC,; SEQ ID NO PAIR 7 CTACCTGGAGTATCATTTTC,, SEQ ID NO GGCTTATAGAGTAGCTTAAAC,; SEQ ID NO PAIR 8 AATATCTTCTCTACCTGG,, SEQ ID NO GATACTTCAAGCTCTATTC,; SEQ ID NO PAIR 9 AGTTTTCATAACAAGTGC,, SEQ ID NO. CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 10: AGTTTTCATAACAAGTGC,, SEQ ID NO GATACTTCAAGCTCTATTC,; SEQ ID NO PAIR 11 CTTCTCTACCTGGAGTATCATTTTC, SEQ ID NO GAGTAGCTTAAACCTAACTTTCCTTG,; SEQ ID NO PAIR 12: CTTCTCTACCTGGAGTATC,, SEQ ID NO GAGTAGCTTAAACCTAACTTTC,; SEQ ID NO PAIR 13 AATATCTTCTCTACCTGG, SEQ ID NO TCATAAGATACTTCAAGC,; SEQ ID NO PAIR 14: AATATCTTCTCTACCTGG, SEQ ID NO 64 CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 15 AATATCTTCTCTACCTGG, SEQ ID NO CATAAGATACTTCAAGCTC,; SEQ ID NO PAIR 16 CTCTACCTGGAGTATCATTTTC, SEQ ID NO GGCTTATAGAGTAGCTTAAACC; SEQ ID NO PAIR 17: CTCTACCTGGAGTATCATTTTC, SEQ ID NO GAGTAGCTTAAACCTAACTTTC; SEQ ID NO PAIR 18: ACCTGGAGTATCATTTTC, SEQ ID NO TCTGGGCTTATAGAGTAG,; SEQ ID NO PAIR 19 CTTCTCTACCTGGAGTATC, SEQ ID NO CTGGGCTTATAGAGTAGC,; SEQ ID NO PAIR 20 CTTCTCTACCTGGAGTATC, SEQ ID NO CTGGGCTTATAGAGTAGC, SEQ ID NO

19. The primer set of claim 18 wherein the primer set is pair 1.

20. The primer set of claim 18 wherein the primer set is pair 3.