WO2015072847A1

WO2015072847A1 - A method of detecting and identifying dengue virus serotype

Info

Publication number: WO2015072847A1
Application number: PCT/MY2014/050007
Authority: WO
Inventors: Sazaly BIN ABU BAKAR; Teoh BOON TEONG
Original assignee: Universiti Malaya
Current assignee: Universiti Malaya
Priority date: 2013-11-14
Filing date: 2014-11-13
Publication date: 2015-05-21
Anticipated expiration: 2016-05-14
Also published as: MY184631A

Abstract

A method of detecting presence of a dengue virus in a biological sample comprises the steps of isolating ribonucleic acid specimen from the biological sample; subjecting the isolated ribonucleic acid specimen to a reverse transcription-loop-mediated isothermal amplification in a single-tube reaction using primers with nucleic acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 throughout a constant temperature involving a forward outer primer, a backward outer primer, a forward inner primer, and a backward inner primer; and ascertaining presence of the dengue virus in the biological sample upon acquiring an amplicon of predetermined sizes. The disclosed method further comprises a primer with nucleic acid sequence as set forth in SEQ ID NO. 9 as the loop primer. The disclosed primers are derived from the 3' untranslated region of the dengue virus genome.

Description

A METHOD OF DETECTING AND IDENTIFYING

DENGUE VIRUS SEROTYPE

Field of Invention

The invention relates to a method for detecting dengue virus in subjects with suspected dengue during the febrile period. More particularly, the invention relates to a method of detecting all dengue virus serotypes through a single-tube reaction using the reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

Background of the Invention

Dengue is caused by dengue virus (DENV), a positive-sense single-stranded ribonucleic acid (RNA) virus that belongs to the genus of Flavivirus. There are at least four antigenically distinct yet closely related DENV serotypes: DENV-1, DENV- 2, DENV-3 and DENV-4; each serotype contains phylogenetically distinct genotypes. Infection with any of the serotypes produces a spectrum of clinical illness ranging from mild dengue fever (DF) to severe and fatal dengue hemorrhagic fever (DHF) and hemorrhagic shock syndrome (DSS). Infection with one serotype leads to lifelong protection against homotypic reinfection but only temporary cross-protection against heterotypic infection. Heterotypic secondary infection is associated with higher risk of contracting dengue hemorrhagic fever and hemorrhagic shock syndrome, possibly through antibody-dependent enhancement, original antigenic sin, cytokine storm or autoimmune response.

Currently, there is no vaccine available for dengue. Hence, preventive measures, such as vector control, are important in minimizing the risk of contracting DENV infection. Dengue-infected viremic individuals are the main source of infectious dengue virus as virus is transmitted following mosquito bites of these viremic individuals. A sensitive, easy to perform and rapid method for detection of DENV in viremic febrile patients is therefore of paramount importance especially for patient management and immediate vector control measures to prevent large-scale epidemic.

Anti-dengue virus immunoglobulin M- (IgM-) and IgG-capture enzyme-linked immunosorbent assays (MAC- and GAC-ELISA) are the most widely used methods to infer DENV infection serologically. However, these assays require second convalescent sera for confirmation of test results. These assays are not applicable during the febrile viremic phase as well owing to significant rise in the antibody titers is yet attained. In addition, antibody cross-reactivity towards other closely related flaviviruses is a common problem in ELISA assays.

Due to the low sensitivity of conventional serological tests, diagnostic tests targeting detection of DENV RNA are deemed more effective during the febrile period. Molecular techniques, such as reverse transcription-polymerase chain reaction (RT- PCR) and real-time quantitative RT-PCR (qRT-PCR) capable of detecting virus genomic RNA sequence in serum samples, have gradually been accepted as new standards over virus isolation for the detection of dengue virus in acute-phase sera. For example, a method of detecting dengue virus using PCR is disclosed in International Patent with publication number WO2013066705. Particularly, the primers and probes disclosed are applicable for single or multiplex polymerase chain reaction which is able to amplify and detect four dengue virus serotypes. However, detection of dengue virus using PCR requires costly PCR instrument and high level of skill that widespread use of PCR-related approach in dengue virus detection may be hard to achieve with lack of skillful lab technicians and expensive equipment.

Further, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is another type of nucleic acid amplification reaction conducted at a constant temperature, thus eliminates the need of expensive thermocycler. Parida et al. (2005) developed a four-tube RT-LAMP assay utilizing serotype-specific primers targeting the proximal half of the 3' untranslated region (UTR) of DENV genome. The four- tube reaction system of Panda et al. requires four separate reactions to be performed. Consequently, it is time-consuming and not cost-effective to be adapted for routine diagnosis. Further, a method of detecting pan-serotype dengue virus using RT-LAMP assay is disclosed in the U.S. Patent No. US2011306036, which employs primers derived from the 5' UTR-capsid region in a single-tube reaction system for the detection of dengue virus. Nevertheless, the targeted capsid gene sequence is relatively less conserved among all four DENV serotypes in comparison to the UTR, resulting in the use of at least 10 primers to achieve the detection coverage for all four DENV serotypes.

In view of the abovementioned shortcomings, a method of detecting dengue virus in a single-tube reaction that maximizes the detection coverage of each primer and uses less number of primer is highly desired. More particularly, such desired method is sensitive and specific towards dengue virus in the febrile period, allowing immediate vector control to minimize the risk of large-scale epidermic.

Summary of the Invention One of the objects of the present invention is to provide a method of detecting dengue virus in a biological sample in a single-tube reaction whereby any of the dengue virus serotypes is detectable.

Further object of the present invention is to offer a method of detecting dengue virus using minimal number of primers. Targeting nucleic acid sequence in the 3' UTR, which is highly conserved among all dengue virus serotypes, maximizes the detection coverage of each primer, and thus reducing the number of primers required for detection of all dengue virus serotypes in a single-tube reaction. Another object of the present invention is to provide a cost-effective and rapid method of detecting dengue virus in a biological sample without the need of resorting to expensive equipment such as a thermocycler and high level of laboratory skills to commence. Still another object of the present invention is to offer a highly sensitive method of detecting dengue virus in a biological sample even when the dengue virus presents in very low copy number.

Further object of the present invention aims to disclose a method of detecting dengue virus with non-detectable cross-reactivity against other closely-related virus such as Japanese encephalitis virus, Chikungunya virus and Sindbis virus.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which the embodiment of the present invention describes a method of detecting presence of a dengue virus in a biological sample comprises the steps of isolating ribonucleic acid specimen from the biological sample; subjecting the isolated ribonucleic acid specimen to a reverse transcription-loop-mediated isothermal amplification in a single-tube reaction using primers with nucleic acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 throughout a constant temperature involving a forward outer primer, a backward outer primer, a forward inner primer, and a backward inner primer; and ascertaining presence of the dengue virus in the biological sample upon acquiring an amplicon of predetermined sizes; wherein the primer SEQ ID NO. 1 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 3 and 4 as the forward outer primer; the primer SEQ ID NO. 2 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 2 as the forward outer primer; the primer SEQ ID NO. 3 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2, and 3 as the backward outer primer; the primer SEQ ID NO. 4 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the backward outer primer; the primer SEQ ID NO. 5 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2 and 3 as the forward inner primer; the primer SEQ ID NO. 6 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the forward inner primer; the primer SEQ ID NO. 7 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2 and 3 as the backward inner primer; and the primer SEQ ID NO. 8 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the backward inner primer.

In a preferred embodiment of the disclosed method, the RNA isolated from a biological sample is amplified at a constant temperature using a set of primers capable of hybridizing onto targeted nucleic acid sequences of dengue virus of any serotype. More preferably, the targeted nucleic acid sequence is derived from the 3' UTR of the dengue virus genome. In the preferred embodiment of the present invention, the set of primers comprises at least one forward outer primer, one backward outer primer, one forward inner primer and one backward inner primer. In another preferred embodiment of the present invention, the set of primers comprises at least one forward outer primer, one backward outer primer, one forward inner primer, one backward inner primer and one loop primer. The loop primer, with nucleic acid sequence as set forth in SEQ ID NO. 9, is capable of hybridizing onto the nucleic acid sequence of dengue virus serotype 1, 2, 3 and 4.

In the preferred embodiment of the present invention, the presence of amplicon is indicated by a fluorescent signal emitted from the reaction mixture used for isothermal DNA amplification reaction. Preferably, the amplicons are digested into fragments of predetermined sizes using restriction enzyme Bcmll. The digested fragments are subjected to agarose gel electrophoresis followed by sequencing. The digested fragments of amplicons of particular sizes indicate the presence of respective dengue virus serotype. The amplicon of dengue virus serotype 1 is digested into fragments of 129 base pair and 233 base pair, the amplicon of dengue virus serotype 2 is digested into fragments of 135 base pair and 231 base pair, the amplicon of dengue virus serotype 3 is digested into fragments of 129 base pair and 231 base pair and the amplicon of dengue virus serotype 4 is digested into fragments of 141 base pair and 231 base pair.

With the aid of the innovative set of primers, the detection of any dengue virus serotype in a biological sample can be conducted within one assay. The innovative primers hybridize to nucleic acid sequences that are conserved in dengue virus genome between generations, thus minimizing the possibility of false negative dengue virus detection caused by mutations at targeted nucleic acid sequences. The innovative primers are highly selective towards dengue virus nucleic acid, thus minimizing the possibility of false positive dengue virus detection caused by closely related viruses such as Japanese encephalitis virus, Chikungunya virus and Sindbis virus.

Brief Description of the Drawings

For the purpose of facilitating an understanding of the present invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the present invention, its construction and operation and many of its advantages would be readily understood and appreciated. Figure 1 shows the alignment between the primers and the dengue virus 3' UTR consensus sequences as well as the orientation of the disclosed primers in 5' to 3' direction as indicated by the arrows.

Figure 2 shows the exemplary visual observations of fluorescence in RT-LAMP reactions under UV light. Figure 3 shows the exemplary results of agarose gel electrophoresis of the undigested and Bcmll digested RT-LAMP amplicons of dengue virus serotype 1, 2, 3 and 4.

Figure 4 illustrates the mean time threshold (Tt) of positivity for RT-LAMP reactions of samples containing ascending predetermined copy of dengue virus RNA. Figure 5 illustrates the number of clinical samples tested positive by RT-LAMP and qRT-PCR.

Figure 6 illustrates the sensitivity of dengue diagnostic methods against laboratory-confirmed dengue samples (n=171).

Detailed Description of the Invention

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the present invention.

The terms "primer", "DNA primer" or "DNA oligonucleotide" are used interchangeably unless otherwise mentioned.

The term "serotype" used herein refers to a group of closely related dengue virus sharing a common set of antigens.

The terms "backward outer primer" or "reverse outer primer" are used interchangeably unless otherwise mentioned whereas the terms "backward inner primer" or "reverse inner primer" are used interchangeably unless otherwise mentioned.

The present invention discloses a method of detecting presence of a dengue virus in a biological sample comprises the steps of isolating ribonucleic acid (RNA) specimen from the biological sample; subjecting the isolated RNA specimen to a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in a single-tube reaction using primers with nucleic acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 throughout a constant temperature involving a forward outer primer, a backward outer primer, a forward inner primer, and a backward inner primer; and ascertaining presence of the dengue virus in the biological sample upon acquiring an amplicon of predetermined sizes; wherein the primer SEQ ID NO. 1 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 3 and 4 as the forward outer primer; the primer SEQ ID NO. 2 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 2 as the forward outer primer; the primer SEQ ID NO. 3 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2, and 3 as the backward outer primer; the primer SEQ ID NO. 4 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the backward outer primer; the primer SEQ ID NO. 5 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2 and 3 as the forward inner primer; the primer SEQ ID NO. 6 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the forward inner primer; the primer SEQ ID NO. 7 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2 and 3 as the backward inner primer; and the primer SEQ ID NO. 8 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the backward inner primer.

As described in the preferred embodiment of the present invention, the dengue virus is categorized into at least four serotypes, namely serotype 1 (DENV-1), serotype 2 (DENV-2), serotype 3 (DENV-3) and serotype 4 (DENV-4). In the preferred embodiment of the present invention, DENV-1 includes but not limited to four strains, which are DENV-1 genotype I, DENV-1 genotype II, DENV-1 genotype III and sylvatic DENV-1; DENV-2 includes but not limited to two strains, which are DENV-2 Asian I and DENV-2 cosmopolitan; DENV-3 includes but not limited to three strains, which are DENV-3 genotype I, DENV-3 genotype II and DENV-3 genotype III; DENV-4 includes but not limited to two strains, which are DENV-4 subgenotype Ila and DENV-4 subgenotype lib. The phrase "biological sample" as used herein encompasses serum as well as animal cell, for example, mosquito cell. According to the preferred embodiment of the present invention, the biological sample is human serum. Initially, RNA specimen from the biological sample is isolated for use in the subsequent nucleic acid amplification reaction. The terms "isolate", "extract" or "purify" are used interchangeably herein unless otherwise mentioned. Preferably, isolation of RNA is conducted using column-based nucleic acid purification. RNA from the biological sample binds specifically to a solid phase in the column and is retained in the column while other components in the biological sample pass through the column. In the preferred embodiment of the present invention, the solid phase is silica. Retained RNA is eluted from the column using a liquid. The preferred liquid used is nuclease- free water. Other commercially available RNA extraction kits employing column- based nucleic acid purification can be used to isolate RNA specimen from the biological sample. Still, other methods including, but not limited to, phenol- chloroform extraction and ethanol precipitation can be used to isolate RNA from the biological sample as well.

Preferably, isolated RNA specimen is subjected to a nucleic acid amplification reaction including reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription-loop-mediated isothermal amplification (RT-LAMP). In the more preferred embodiment of the present invention, the nucleic acid amplification reaction is a RT-LAMP. As embodied herein, the present invention, the method of detecting dengue virus in a single-tube reaction, is characterized by the amplification of RNA specimen using a set of primers highly selective towards all four dengue virus serotypes. Preferably, the dengue virus nucleic acid sequences recognizable by the primers are derived from common regions in the 3' UTR of dengue virus genome shared between four dengue virus serotypes. The alignment between the primers and the 3' UTR of four dengue virus serotypes are shown in Figure 1 where the arrows indicate the orientation of primers in the direction from 5' to 3'. More preferably, the disclosed primers recognize the nucleic acid sequences in the 3' UTR regions of dengue virus serotypes including, but not limited to, DENV-1 genotype I, DENV-1 genotype II, DENV-1 genotype III, sylvatic DENV-1, DENV-2 Asian I, DENV-2 cosmopolitan, DENV-3 genotype I, DENV-3 genotype II, DENV-3 genotype III, DENV-4 subgenotype Ila and DENV-4 subgenotype lib. In the preferred embodiment of the present invention, the set of primers recognizes six distinct regions of the dengue virus nucleic acid sequence. The set of primers used to amplify a targeted region of the RNA comprises eight primers; SEQ ID NO. 1 and SEQ ID NO. 2 are forward outer primers, SEQ ID NO. 3 and SEQ ID NO. 4 are backward outer primers, SEQ ID NO. 5 and SEQ ID NO. 6 are forward inner primers, and SEQ ID NO. 7 and SEQ ID NO. 8 are backward inner primers. The nucleic acid sequences of primers SEQ ID NO. 1 to 8 are shown in Table 1 in Example 2 where a RT-LAMP assay using this set of primers is detailed.

In the preferred embodiment of the present invention, the forward outer primer and backward outer primer recognize one distinct region of the dengue virus nucleic acid sequence, respectively. The forward inner primer and backward inner primer recognize two distinct regions of the dengue virus nucleic acid sequence, respectively, whereby the 3' end of the inner primer hybridizes to a region located upstream of the region hybridized by the outer primer and the 5' end of the inner primer has nucleic acid sequence identical to the nucleic acid sequence of a region located upstream of the region hybridized by the 3' end of the inner primer. With the use of inner primers, amplicons produced contain multiple repeats of targeted nucleic acid sequence, each of which connected by stem-loops. In another preferred embodiment of the present invention, the set of primers recognizes seven distinct regions of the dengue virus nucleic acid sequence. The set of primers used to amplify a targeted region of the RNA comprises nine primers; SEQ ID NO. 1 and SEQ ID NO. 2 are forward outer primers, SEQ ID NO. 3 and SEQ ID NO. 4 are backward outer primers, SEQ ID NO. 5 and SEQ ID NO. 6 are forward inner primers, SEQ ID NO. 7 and SEQ ID NO. 8 are backward inner primers and SEQ ID NO. 9 is loop primer. The nucleic acid sequences of primers SEQ ID NO. 1 to 9 are shown in Table 2 in Example 3 where a RT-LAMP assay using this set of primers is detailed. In another preferred embodiment of the present invention, the loop primer refers to a backward loop primer recognizing a region located in between the regions hybridized by the 3' end and 5' end of the backward inner primer. Loop primer accelerates the reaction of RT-LAMP by increasing the number of starting points of DNA amplification. Particularly, it hybridizes to the stem-loops of an amplicon that are not hybridized by the inner primers. With the aid of the loop primer, a RT-LAMP reaction produces predetermined copies of amplicons in a relatively shorter reaction time.

In the preferred embodiment of the present invention, reverse transcriptase is used to reverse transcribe dengue virus RNA into DNA. Reverse transcriptase binds to the dengue virus RNA specimen in the biological sample upon hybridization of inner primer to the RNA specimen and synthesizes a complementary deoxyribonucleic acid (cDNA) strand of the virus RNA template. The synthesized cDNA is displaced and released from the RNA template when an outer primer hybridizes to the same RNA template and reverse transcriptase begins synthesizing another cDNA strand from the RNA template. Stem-loop forms at one end of the released cDNA due to the incorporation of inner primer whose DNA sequence at 5' end is complementary to an internal region of the cDNA. Another inner primer hybridizes to the released cDNA on the unlooped end. A DNA polymerase begins to incorporate complementary nucleotides at the 3' end of the inner primer in the direction of 5' to 3'. Preferably, a DNA polymerase with strand displacement activity is used in the nucleic acid amplification reaction so that even a double-stranded DNA is capable to serve as template for nucleic acid amplification without being heat-denatured into single- stranded DNA prior to amplification. The newly synthesized DNA strand is displaced from the cDNA template by the DNA polymerase when an outer primer binds the template and DNA polymerase begins to incorporate complementary nucleotides at the 3' end of the outer primer in the direction of 5' to 3'. The displaced DNA strand forms two stem-loops at opposite ends due to the presence of two inner primers whose DNA sequences at their 5' ends are complementary to two internal regions, respectively, of the displaced DNA strand. This dumbbell-like structure allows for continuous nucleic acid amplification whereby amplicons containing various copy number of targeted nucleic acid sequence are produced. In another embodiment of the present invention, loop primers hybridizes to the stem-loops of the amplicons to which inner primers do not hybridize and DNA polymerase incorporates complementary nucleotide at the 3' end of the loop primer in the direction of 5' to 3'.

If the isolated RNA specimen does not contain any of the region recognizable by any of the primer, nucleic acid amplification will not happen. If the isolated RNA specimen contains at least one but not all of the regions recognizable by the forward outer primer, backward outer primer, forward inner primer and backward inner primer, primer extension occurs but RT-LAMP cycling amplification will not occur. RT-LAMP cycling amplification occurs only when the isolated RNA specimen contains all the regions recognizable by the forward outer primer, backward outer primer, forward inner primer and backward inner primer. In the preferred embodiment of the present invention, the presence of amplicons is detected by observing the turbidity of the reaction mix. The turbidity of the reaction mix is contributed by the presence of magnesium pyrophosphate produced during the nucleic acid amplification reaction. Preferably, during the nucleic acid amplification reaction, the turbidity of the reaction mixture is measured at 650 nm using a spectrophotometer whereby the threshold value of turbidity is set at 0.06-0.08 absorbance unit. In the another preferred embodiment of the present invention, the presence of amplicons is also visually detected by using a reagent for nucleic acid amplification whereby the color of the reagent changes and fluorescent signal is emitted upon successful nucleic acid amplification. Preferably, the reagent contains calcein. Pyrophosphate produced during nucleic acid amplification reaction, and magnesium ions stimulate calcein to emit fluorescence and intensify the fluorescence, respectively. In the preferred embodiment of the present invention, a positive detection of amplicons refers to a reaction mixture that changes its color from orange to green and fluoresces under UV light. On the other hand, a negative detection of amplicons refers to a reaction mixture that remains orange in color and does not fluoresces under UV light. Figure 3 shows the visual observation of positive and negative detection of amplicons based on fluorescence emitted under UV light.

In another embodiment of the present invention, the presence of amplicons is detected by subjecting the amplicons to an agarose gel electrophoresis. The formation of a ladder-like pattern on agarose gel indicates the presence of amplicons as each of which contains various copy number of targeted nucleic acid sequence.

In another embodiment of the present invention, the serotype of the amplicons of the dengue virus present in the biological sample is identified by subjecting the amplicons to an agarose gel electrophoresis and sequencing. Methods including, but not limited to, immunostaining such as enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) are also used to identify the serotype of dengue virus. In another embodiment of the present invention, the amplicons from RT-LAMP are subjected to a restriction enzyme digestion prior to agarose gel electrophoresis. Preferably, the restriction enzyme is Banll which is capable of carrying out a single site restriction enzyme digestion on the amplicons. Amplicons that are digested into two fragments are likely to be the amplicons of dengue virus RNA.

Digested amplicons are then subjected to an agarose gel electrophoresis. In another embodiment of the present invention, the agarose gel is stained with a nucleic acid gel stain. Preferably, the nucleic acid gel stain is GelRed™ . However, nucleic acid gel stains including, but not limited to, ethidium bromide, Gel Green™, Gel Star™, SYBR® Gold, SYBR® Green I, SYBR® Safe, Blue View™, DAPI, acridine orange and methylene blue can be used to stain the agarose gel. In another embodiment of the present invention, the stained gel is visualized using a UV transilluminator. Figure 3 shows the image of a stained agarose gel after running a gel electrophoresis using undigested and Banll digested amplicons of dengue virus serotypes 1 to 4.

In another embodiment of the present invention, the digested amplicons are subjected to sequencing. Preferably, loop primer with nucleic acid sequence SEQ ID NO. 9 is used as DNA probe in sequencing the digested amplicons. However, DNA probes including, but not limited to, fragments derived from loop primer can also be used in sequencing the digested amplicons. In another embodiment of the present invention, the Banll digested fragments of the amplicons of DENV-1 are having the size of 129 base pair (bp) and 233 bp; the Banll digested fragments of the amplicons of DENV-2 are having the size of 135 bp and 231 bp; the Banll digested fragments of the amplicons of DENV-3 are having the size of 129 bp and 231 bp; the Banll digested fragments of the amplicons of DENV-4 are having the size of 141 bp and 231 bp.

In another embodiment of the present invention, the sensitivity of RT-LAMP assay is measured using a panel of samples with ascending predetermined copy of dengue virus RNA template. The dengue virus RNA copy number is quantitated using qRT- PCR. Particularly, a fluorescently-labeled oligonucleotide probe capable of hybridizing onto the nucleic acid sequence of the dengue virus is used in the qRT- PCR reaction. The probe can be labeled with fluorescent dye including, but not limited to, 6-FAM^®, HEX^®, NED^®, JOE™, TET™, TAMRA™, Cy3^®, Cy5^®, Cy5.5^®, Cal Fluor® Gold 540, Cal Fluor Orange 560, Cal Fluor Red 590, Quasar^® 570, Quasar 670, ROX™, and Texas Red^® A commercially available kit for qRT-PCR of dengue virus can be used. Figure 4 shows the time threshold of positivity for RT-LAMP assays of a panel of samples containing ascending predetermined copy of dengue virus RNA. The detection limit of the disclosed RT-LAMP assay is determined from the number of positive detection of DENV RNA in each positivity test. The phrase "detection limit" used herein refers to the lowest amount of template detectable with near 100% reproducibility throughout multiple assays.

In another embodiment of the invention, the test results for detecting the presence of dengue virus RNA or anti-dengue antibody in a biological sample using any one or combination of RT-LAMP, qRT-PCR, and ELISA are compared as shown in Example 6. Two separate ELISA assays are conducted to detect the presence of anti-dengue IgM and anti-dengue IgG antibodies, respectively. Commercially available ELISA kits for detection of anti-dengue IgM and IgG antibodies can be used. Positive detection of dengue virus RNA and/or anti-dengue IgM infers acute dengue virus infection whereas positive detection of anti-dengue IgG but negative detection of dengue virus RNA and anti-dengue IgM infers past dengue virus infection. In another embodiment of the present invention, kappa value (κ) measures the degree of agreement between the test results of RT-LAMP and qRT-PCR. Figure 5, Table 2 and Table 3 show the comparison of the test results of RT-LAMP, qRT-PCR and ELISA (IgM and IgG). Figure 6 shows the comparison of the test results of RT-LAMP, qRT- PCR, ELISA and the combination of any two of them.

As described in the foregoing description, the set of primers can be used to specifically identify the presence of dengue virus in the biological sample with non- detectable cross-reactivity towards closely related arboviruses such as Japanese encephalitis virus, Chikungunya virus and Sindbis virus. With the use of the set of primers, any of the four dengue virus serotypes can be detected within a single reaction tube. The method of detecting dengue virus by RT-LAMP reaction is specific, sensitive, simple, inexpensive and quick, thus can be employed as routine clinical diagnostic tool of dengue virus infection.

Example

An example is provided below to illustrate different aspects and embodiments of the present invention. The example is not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1 Isolation of RNA specimen

A total of 305 serum samples from patients of University Malaya Medical Centre clinically suspected with dengue virus infection are obtained within two months. Serving as sources of reference dengue virus RNA, C6/36 Aedes albopictus mosquito cells are inoculated with 11 different strains of dengue viruses and cultured for one week. The viruses, including DENV-1 genotype I, DENV-1 genotype II, DENV-1 genotype III, sylvatic DENV-1, DENV-2 Asian I, DENV-2 cosmopolitan, DENV-3 genotype I, DENV-3 genotype II, DENV-3 genotype III, DENV-4 subgenotype Ila and DENV-4 subgenotype lib, are archived in the University Malaya Medical Centre (UMMC) Diagnostic Virology Laboratory repository.

Total RNA is extracted from 140 μΙ_, of patient serum or the supernatant of dengue virus-infected mosquito cell culture using QIAamp virus RNA Mini Kit, following the manufacturer's protocol. The RNA retained in the spin column is eluted with 60 μΙ_, of nuclease-free water and stored at -80 °C until needed. Example 2 RT-LAMP assay (I)

The RT-LAMP is performed in a final reaction volume of 25 [iL using a Loopamp RNA Amplification Kit (Eiken Chemical Co. Ltd., Japan) following the manufacturer's protocol. The nucleic acid sequences of primers used in RT-LAMP are shown in Table 1. 20 pmol each of primers SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8; 2.5 pmol each of primers SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4; 1 [iL of Fluorescent Detection Reagent (Eiken Chemical Co. Ltd., Japan); and 5 [iL of extracted RNA specimen are added to the reaction mixture. A positive control using 1000 copies (determined by qRT-PCR) of dengue virus RNA extracted from infected cell culture supernatant and a negative control, nuclease-free water, are included in each run of RT-LAMP reaction.

The RT-LAMP reaction mixtures are incubated at 63 °C for 80 min and inactivated at 80 °C for 5 min in LA-500 Loopamp real-time turbidimeter (Eiken Chemical Co. Ltd., Japan). The turbidity of RT-LAMP reaction mixture is spectrophotometrically recorded at 650 nm every 6 s. The threshold time (Tt) value for positivity by RT- LAMP is determined when the turbidity increases above the threshold value, 0.07 absorbance units.

The color change of the reaction mixture under white light and UV light is observed. The reaction mixture becomes green in color and fluoresces under UV light when RT- LAMP takes place. In contrast, the reaction mixture remains orange in color and does not fluoresce under UV light when RT-LAMP does not takes place.

Figure 2 shows the illustration of color change of reaction mixture under UV light in the presence and absence of RT-LAMP. Tube 1 serves as the negative control; tube 2- 5 contains RNA sample from dengue virus DENV-1 genotype I, II, III and sylvatic, respectively; tube 6-7 contains RNA sample from dengue virus DENV-2 Asian I and cosmopolitan, respectively; tube 8-10 contains RNA sample from dengue virus DENV-3 genotype I, II and III, respectively; tube 11-12 contains RNA sample from dengue virus DENV-4 subgenotype Ila and lib, respectively; tube 13-15 contains RNA sample from Japanese encephalitis virus (JEV), Chikungunya virus (CHTKV) and Sindbis virus (SINV), respectively. As shown in Figure 2, absence of RT-LAMP reactions in tube 13-15 indicates that the disclosed primers do not cross-react with the closely-related arboviruses JEV, CHTKV and SINV.

Example 3 RT-LAMP assay (II)

The RT-LAMP assay is performed as previously described in Example 2 using a 25 μΕ reaction mixture containing 20 pmol each of primers SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8; 2.5 pmol each of primers SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4; 20 pmol of primer SEQ ID NO. 9; 1 of Fluorescent Detection Reagent (Eiken Chemical Co. Ltd., Japan); and 5 μΕ of extracted RNA specimen. The nucleic acid sequences of primers used in this RT- LAMP assay are shown in Table 2. Table 2

SEQ Primer Dengue Sequence (5'→ 3')

ID NO. type^* serotype(s)

1 F3 1, 3, 4 CAAACCGTGCTGCCTGT

2 F3 2 TGAGTAAACTATGCAGCCTGT

3 B3 1, 2, 3 ACCTGTTGATTCAACAGCACC

4 B3 4 ACCTGTTGGATCAACAACACC

5 FIP 1, 2, 3 AGGGGTCTCCTCTAACCRCTAGTCTTTCAAACCRTGGAAGCTGTACGC

6 FIP 4 AGGGGTCTCCTCTAACCRCTAGTCTTTTTTGCCACGGAAGCTGTACGC

7 BIP 1, 2, 3 ACAGCATATTGACGCTGGGARAGACGTTCTGTGCCTGGAATGATGCTG

8 BIP 4 ACAGCATATTGACGCTGGGARAGACGCTCTGTGCCTGGATTGATGTTG

9 BLP 1, 2, 3, 4 CAGAGATCCTGCTGTCTC

^* F3= Forward outer primer; B3=Backward outer primer; FIP=Forward inner primer; BIP=Backward inner primer; BLP=Backward loop primer.

Example 4 Identification of dengue virus serotype

RT-LAMP amplicons of DENV-1, DENV-2, DENV-3 and DENV-4 are digested with restriction enzyme Banll (New England Biolabs Inc., US). The restriction enzyme digestion is performed in a final volume of 50 μΐ. containing 5 μΐ. of lOx NEBuffer 4, 1 μΐ. of Banll, 43 μΐ. of nuclease-free water, and 1 μί of RT-LAMP amplicons. The reactions are incubated at 37 °C overnight. The undigested and digested RT-LAMP amplicons are electrophoresed on a 2% agarose gel in Tris-acetate-EDTA buffer. The gel is stained with GelRed™ (Biotium Inc., US) and visualized using a Gel Doc 2000 (Bio-Rad Laboratories, Inc., US). Figure 3 shows the image of agarose gel containing undigested and Banll digested amplicons of DENV-1, DENV-2, DENV-3 and DENV-4. Ladder-like pattern formed on the agarose gel indicates the presence of amplicons of dengue virus. As shown in Figure 3, lane M contains 100-base pair (bp) plus DNA ladder; lane 1 contains undigested amplicons of dengue virus serotype 1; lane 2 contains fragments of Banll digested amplicons of dengue virus serotype 1 with the sizes of 129 bp and 233 bp; lane 3 contains undigested amplicons of dengue virus serotype 2; lane 4 contains fragments of Banll digested amplicons of dengue virus serotype 2 with the sizes of 135 bp and 231 bp; lane 5 contains undigested amplicons of dengue virus serotype 3; lane 6 contains fragments of Banll digested amplicons of dengue virus serotype 3 with the sizes of 129 bp and 231 bp; lane 7 contains undigested amplicons of dengue virus serotype 4; lane 8 contains fragments of Banll digested amplicons of dengue virus serotype 4 with the sizes of 141 bp and 231 bp. The digested amplicons are sequenced using the primer SEQ ID NO. 9. The sizes of the digested amplicons are in agreement with the expected size for each serotype. Banll digestion of amplicons of DENV-1, DENV-2, DENV-3 and DENV-4 produces two digested fragments of particular sizes for each dengue virus serotype, indicating that the disclosed primers are specific towards 3' UTR of DENV genome. Example 5 Assessment of sensitivity of RT-LAMP assay

Twelve RT-LAMP assays are performed using 10, 60, 100 and 1000 copies of dengue virus RNA, respectively. The dengue virus RNA is extracted from the infected cell culture supernatant and the copy number of virus RNA is quantitated using the genesig Real-Time qRT-PCR DENV detection kit (PrimerDesign Ltd., UK). The qRT- PCR assay standard plot, ranged from 10 to 10⁶ RNA copies, is made by preparing a 10-fold serial dilution of the genesig DENV RNA standard. The genesig DENV RNA standard is a synthetic RNA template with known copy number. The qRT-PCR is performed in a final volume of 20 uL containing 10 uL of real time master mix, 1 of probe/primer mix, 4 uL of nuclease-free water, and 5 μΐ_^ of diluted RNA. Quantitative PCR measurement is performed using StepOnePlus real time PCR system (Applied Biosystems, USA) according to the following condition: 10 min at 55 °C, 8 min at 95 °C followed by 95 cycles of amplification (10 s at 95 °C, 60 s at 60 °C). Raw data is analyzed with StepOne Software v2.2.1 to determine copy number based on the threshold cycles (Ct). The efficiency of the qRT-PCR is measured from the slope of standard curve.

Figure 4 shows the time threshold (Tt) of positivity for RT-LAMP assays of 10, 60, 100 and 1000 copies of dengue virus RNA. Each positivity test is repeated for 12 times. The number of positive detection by RT-LAMP assays (n=12) for the DENV RNA with copy numbers of 1000, 100, 60 and 10 are 100% (12/12), 100% (12/12), 75% (9/12) and 25% (3/12), respectively, with the mean time threshold (Tt) of 46.97 ± 2.28 min, 53.67 ± 1.77 min, 53.78 ± 2.89 min, 53.28 ± 5.04 min, respectively. RT- LAMP assay can detect up to 10 copies of DENV RNA but at least 100 copies of DENV RNA are required to achieve near 100% reproducible positive DENV detection by RT-LAMP. As suggested by the results, RT-LAMP assay using the disclosed primers has a detection limit of at least 100 copies of viral RNA. As reported by Levi et al., 2007 and Kong et al., 2006, the viral load is inversely proportional to the antibody titer according to the day of onset of fever. Low viral load (<100 viruses) is usually accompanied with the rise of antibody titer. The detection of DENV using RT-LAMP regardless of the viral load could be completed within 1 hour making the assay a rapid tool for early dengue diagnosis.

Example 6 Evaluation of RT-LAMP assay

RT-LAMP assays are performed using RNA specimen isolated from a total of 305 serum samples. 23 of the 305 samples are collected 1 to 5 days after the collection of 282 serum samples. qRT-PCR assays are served as reference assays for detection of dengue virus RNA in the sera. Presence of anti-dengue IgM in the sera is screened using the SD Dengue IgM Capture ELISA Kit (Standard Diagnosis Inc., Korea). IgM- negative sera are furthered screened for the presence of anti-dengue IgG using the SD Dengue IgG capture ELISA Kit (Standard Diagnosis Inc., Korea). The test results of RT-LAMP, qRT-PCR and ELISA are then compared. Positive detection of anti-dengue IgM by ELISA and/or the presence of dengue virus RNA detected by qRT-PCR assay affirm acute dengue virus infection. Past dengue virus infection is indicated by presence of only anti-dengue IgG in the serum.

Table 3 shows the comparison of test results of RT-LAMP, qRT-PCR and ELISA for detection of dengue virus in serum samples. As shown in table 3, acute dengue infection is confirmed in 171 (56.1%) by either qRT-PCR or dengue IgM ELISA, or both. 5 out of the 171 samples are identified as secondary dengue infection as virus RNA and dengue-specific IgG are detected in the absence of anti-dengue IgM. 7 serum samples are identified as past dengue infection as only anti-dengue IgG is tested positive.

Table 4 shows the comparison between the diagnostic performance of RT-LAMP and qRT-PCR in serum samples. The RT-LAMP assay detects DENV genome in 74 of 171 (43.3 %) of the acute dengue samples compared to 80 of 171 (46.8%) by qRT-PCR assay. These two methods, however, show high concordance with kappa value of 0.939 (p<0.001).

Figure 5 shows the comparison of test results between RT-LAMP and qRT-PCR. All of these samples contain less than 50 copies of RNA per reaction. As shown in figure 5, 6 additional samples are tested positive by qRT-PCR. 1 sample is considered as false positive as it is tested positive by RT-LAMP but negative by qRT-PCR or ELISA. The performance of the RT-LAMP assay is validated by simultaneous testing of the samples using real-time qRT-PCR, which is known as the most sensitive and specific method for the detection of viral RNA. As shown in tables 3 and 4, both the RT- LAMP and qRT-PCR assays show comparable sensitivity for the detection of DENV in patients' sera (κ=0.939) even though the sensitivity of RT-LAMP is slightly lower than that of qRT-PCR. As shown in figure 5, the RT-LAMP assay gives possibly six false negative and one false positive results. The false negative detection could be caused by low viral load, below the detection limit of RT-LAMP. However, this could also reflect false positive detection of the qRT-PCR. This could be verified by culturing the samples for virus isolation. Similarly, the false positive RT-LAMP result could be a true positive but need to be verified by virus isolation.

The comparison of the test results of RT-LAMP, qRT-PCR, ELISA and the combination of any two of them is shown in figure 6. The combination of RT-LAMP with the anti-dengue IgM and IgG ELISA results in a significant increase (p<0.001) in sensitivity to 97.7% (167 of 171) in comparison to using anti-dengue IgM and IgG ELISA alone which have sensitivity of 70.8% (121 of 171).

Currently, serological assay, the anti-dengue IgM and IgG ELISA, is the most common method used to confirm dengue infection. As shown in figure 6, 46.8% (80 of 171) of serum samples collected from febrile dengue patients indicates that the patients are viremic with the absence of IgM or IgG. The RT-LAMP or qRT-PCR, when used in combination with ELISA, increases the diagnostic coverage of febrile dengue patient to more than 97%. The early detection of viremic individuals allows for immediate intervention to prevent further spread of dengue by encouraging the patient to take precaution to prevent from mosquito bites. In addition, it allows the patient to have sufficient warning to immediately return to the hospital if warning signs of impending severe dengue develops.

References

1. Panda, M; Horioke, K; Ishida, H; Dash, P.K.; Saxena, P; Jana, A.M.; Islam, M.A.; Inoue, S; Hosaka, N.; Morita, K. 2005. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay. Journal of Clinical Microbiology 43 (6): 2895-2903.

2. Levi, J.E.; Tateno, A.F.; Machado, A.F.; Ramalho, D.C.; de Souza, V.A.; Guilarde, A.O.; de Rezende Feres, V.C.; Martelli, CM.; Turchi, M.D.; Siqueira, J.B.Jr; Pannuti, C.S. 2007. Evaluation of a commercial real-time PCR kit for detection of dengue virus in samples collected during an outbreak in Goiania, Central Brazil, in 2005. Journal of Clinical Microbiology 45 (6): 1893-1897.

3. Kong, Y.Y.; Thay, C.H.; Tin, T.C.; Devi, S. 2006. Rapid detection, serotyping and quantitation of dengue viruses by Taqman real-time one-step RT-PCR. Journal of Virological Methods 138 (1-2): 123-130.

Claims

1. A method of detecting presence of a dengue virus in a biological sample comprising the steps of

isolating ribonucleic acid specimen from the biological sample;

subjecting the isolated ribonucleic acid specimen to a reverse transcription-loop- mediated isothermal amplification in a single-tube reaction using primers with nucleic acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 throughout a constant temperature involving a forward outer primer, a backward outer primer, a forward inner primer, and a backward inner primer; and

ascertaining presence of the dengue virus in the biological sample upon acquiring an amplicon of predetermined sizes;

wherein the primer SEQ ID NO. 1 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 3 and 4 as the forward outer primer; the primer SEQ ID NO. 2 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 2 as the forward outer primer; the primer SEQ ID NO. 3 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2, and 3 as the backward outer primer; the primer SEQ ID NO. 4 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the backward outer primer; the primer SEQ ID NO. 5 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2 and 3 as the forward inner primer; the primer SEQ ID NO. 6 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the forward inner primer; the primer SEQ ID NO. 7 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotypes 1, 2 and 3 as the backward inner primer; and the primer SEQ ID NO. 8 is capable of hybridizing onto ribonucleic acid specimen of dengue virus serotype 4 as the backward inner primer.

2. A method according to claim 1 further comprising a primer with nucleic acid sequence as set forth in SEQ ID NO. 9 that is capable of hybridizing onto ribonucleic acid specimen of dengue virus 1, 2, 3 and 4 as the loop primer.

3. A method according to claim 1 further comprising the step of identifying the serotype of dengue virus present in the biological sample by subjecting the amplicon for a gel electrophoresis or sequencing.

4. A method according to claim 1 further comprising a step of digesting the amplicon using a restriction enzyme to produce a plurality of digested fragments of predetermined sizes; wherein the amplicon of dengue virus serotype 1 is digested into fragments of 129 base pair and 233 base pair, the amplicon of dengue virus serotype 2 is digested into fragments of 135 base pair and 231 base pair, the amplicon of dengue virus serotype 3 is digested into fragments of 129 base pair and 231 base pair and the amplicon of dengue virus serotype 4 is digested into fragments of 141 base pair and 231 base pair.

5. A method according to claim 4 further comprising a step of sequencing nucleotides of the digested fragments for identifying the serotype of dengue virus present in the biological sample.

6. A method according to claim 4, wherein the restriction enzyme is Banll.

7. A method according to claim 1, wherein the biological sample is a serum or an animal cell.

8. A method according to claim 1, wherein the nucleic acid sequence amplified is derived from a 3' untranslated region of a genome of the dengue virus.