ASSAY TO INVESTIGATE SPECIFIC SNPS IN MULTIPLE GENES AS HOST GENETIC RISK FACTORS FOR ENTERO VIRU S71 INFECTION SEVERITY

TECHNICAL FIELD:

[001] The present invention relates to molecular biology. Specifically, the present disclosure is related to development and designing Single Nucleotide Polymorphism (SNP) primers along with corresponding thirty Polymerase Chain Reaction (PCR) Primers for the SNP detection assay of SNPs related to genetic susceptibility for Enterovirus A71 (EV-A71).

BACKGROUND ART:

[002] Enterovirus A71 belongs to the species Enterovirus A of the genus Enterovirus in virus family Picornaviridae. Nearly 110 different enteroviruses infecting humans have been identified and classified into 4 species Enterovirus A to Enterovirus D. The enterovirus is composed of one molecule of single stranded positive sense RNA as its genome and 4 structural proteins (VP1 to VP4) forming its capsid. As a group enterovirus cause a variety of diseases of humans ranging from mild diarrhea to severe gastroenteritis, aseptic meningitis, encephalitis, acute flaccid paralysis, myocarditis, juvenile diabetes, hand foot and mouth disease and acute hemorrhagic conjunctivitis among others. Poliovirus was the most dreadful of the human enteroviruses. Since antiquity poliovirus has permanently disabled children by causing paralytic poliomyelitis. In 1988, World Health Organization (WHO) committed all the nations to eradicate polio. When WHO polio program began, there were nearly 350000 polio cases used to be reported annually in the world. Polio program is the 2 ^nd largest global effort to eradicate a disease/ infections. As of today, polio has been eradicated from most of the world except Pakistan and Afghanistan. Polio eradication is the most successful public health program of the recent times.

[003] The next important Enterovirus is EV-A71. EV-A71 has now been recognized as an infection of global public health concern. EV-A71 was discovered in 1969 in USA. EV-A71 was responsible for severe encephalitis cases in European Countries. In 1975, EV-A71 caused an outbreak of 320 cases of aseptic meningitis, encephalitis and polio like paralysis. EV-A71 is known to cause outbreaks of Hand Foot and Mouth Disease (HFMD), aseptic meningitis and encephalitis with severe neurological disease. In 1997, EV-A71 was responsible for very large epidemic of severe HFMD followed by neurological disease, pulmonary edema and death in Sarawak, Malaysia. In 1998, more than 198000 cases of EV-A71 HFMD were reported in Taiwan. There were a large number of deaths due to neurological complication of EV-A71. From here onwards, EV- A71 has been causing HFMD outbreak in Asia Pacific countries including China, Taiwan, Thailand, Singapore, Malaysia, Japan, Vietnam, and Australia. In recent years (2005 onwards) China has experienced HFMD outbreaks annually. Between 2008 and 2012 about 82000 serious cases of HFMD and more than 2457 deaths have been reported. In the first quarter of 2018, about 140000 HFMD were reported.

[004] EV-A71 has been reported in most countries, though Asians appear to be the most vulnerable populations. Pathogenesis studies have cited single nucleotide polymorphisms (SNPs) in about 12 genes that likely influence EV-A71 susceptibility, severity and treatment prognosis (Table 1). In India, EV-A71 was first suspected in an outbreak of HFMD in Calicut (serological evidence only). The first EV-A71 isolation in India was reported from the stool samples of a case of AFP from Uttar Pradesh in 2000. It is now confirmed that EV-A71 is endemic in the country. However, unlike other Asian countries no clustered cases of AFP, aseptic meningitis and/or encephalitis caused by EV- A71 or outbreaks of EV-A71 HFMD have been recorded in the country. These observations are suggestive of genetic basis for susceptibility to EV-A71 infection.

[005] Genomic characterization of EV-A71 strains isolated globally has revealed 7 genotypes designated as EV-A71 genotypes A through G. Genotype A represents the prototype strain BrCr isolated in the US. Genotype A is not in circulation though 2 isolates were reported in China recently. Genotype B consists of sub-genotypes B0-B5. This was considered the most pathogenic EV-A71. Genotype C consists of sub-genotypes C1-C4. Genotype D and genotype G are indigenous and genotype D is the predominant type in India. Genotypes D and G has not been reported from any other country. Genotypes E and F were reported more recently from Africa.

[006] In recent years, interest has grown to investigate genetic predisposal to EV-A71 disease and its severity. Researchers have described genotypes (SNP) in host genome that may confer susceptibility to EV-A71 infections. Fiterature search showed 15 SNPs in 12 genes responsible for EV-A71 susceptibility. The 12 genes are listed in Table 1. These SNPs are recognized as genetic susceptibility markers of EV-A71.

Table 1: SNP positions and name of genes from literature:

[007] The prior arts have been referred in separate reference column, which may be taken into confidence. In addition, a wide variety of techniques have been developed for SNP detection and analysis. Regarding said technique and its positive exploitation, US 5,858,659; US 5,633,134; US 5,719,028; WO98/30717; WO97/10366; W098/44157; WO98/20165; WO95/12607 and WO98/30883 may be referred. In addition, ligase based methods are described by W097/31256 and Chen et al. Genome Res. 1998 ;8(5):549-56; mass-spectroscopy-based methods by W098/12355, W098/14616 and Ross et al. (1997) Anal Chem. 15, 4197-202; PCR-based methods by Hauser, et al. (1998) Plant J. 16,117- 25; exonuclease-based methods by Mundy vide US 4,656,127; dideoxynucleotide-based methods by W091/02087; Genetic Bit Analysis or GBA™ by W092/15712; Oligonucleotide Ligation Assays or OLAs by Landegren et al.(l988) Science 241:1077- 1080 and Nickerson et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927; and primer-guided nucleotide incorporation procedures by Prezant et al.(l992) Hum. Mutat. 1:159-164; Ugozzoli et al.(l992) GATA 9:107-112; Nyreen et al. (1993) Anal. Biochem. 208:171-175; US 6410231 and US20080070792 discloses several SNP Protocols.

[008] It is noteworthy that there is no assay available as of to-date to investigate multiple genetic susceptibility markers (SNPs) to EV-A71 which can be used for rapid screening of patients and populations.

[009] The crux of the invention lies in designing the new and novel PCR primers, novel SNP primers and the novel multiplexed SNP assays using these primer sets for detecting genetic markers of susceptibility to EV-A71 which give exceptionally good and dependable results with high accuracy.

OBJECTIVE OF THE INVENTION:

[0010] One objective of the invention is to provide Single Nucleotide Polymorphism (SNP) primers along with corresponding thirty Polymerase Chain Reaction (PCR) Primers for the SNP detection assay of SNPs related to genetic susceptibility for Enterovirus A71 (EV-A71).

[0011] In one other objective, the invention provides an assay for investigation of specific multiples SNPs as host genetic risk markers of susceptibility to EV-A71 disease. The said invention is universally applicable.

[0012] Another objective is to analyze EV-A71 specific host genetic risk SNP profiles using indigenously developed multiplexed SNP assay.

[0013] Another objective is to design appropriate SNP primers and corresponding PCR primers for appropriate running of assay. [0014] Yet another objective is to compare prevalence of host genetic risk markers universally with published data from countries experiencing frequent EV-A71 epidemics.

[0015] Still another objective is to determine population susceptibility to EV-A71 disease as a public health issue universally.

[0016] Further object is to construct panels of“SNP profiles” for future use as internal reference standards.

[0017] Yet another object is to develop an efficient and dependable assay for detection of SNPs related to genetic susceptibility for Enterovirus A71 (EV-A71).

[0018] Yet another object of the present invention is to develop a kit comprising of Single Nucleotide Polymorphism (SNP) primers along with corresponding thirty Polymerase Chain Reaction (PCR) Primers.

SUMMARY OF THE INVENTION

[0019] The present invention discloses primers for an Assay to investigate multiple SNPs as host genetic risk markers for EV-A71, said primers constitutes of both polymerase chain reaction (PCR) primers required for due amplification of genetic materials; the said PCR primers constitute of Sequence ID Nos.: SJ201701 to SJ201730, and SNP primers for detecting 15 single nucleotide polymorphisms (SNPs) in 12 different genes. The SNP primers constitute of sequence ID Nos.: SJ201731 to SJ2017 45. Primers are listed hereinbelow. [0020] Multiplex PCR Primers:

Sr No. 1. Seq ID SJ201701 has been developed based upon rsl800896SNP Sr No. 2. Seq ID SJ201702 has been developed based upon rsl800896SNP Sr No. 3. Seq ID SJ201703 has been developed based upon rs4l284767SNP Sr No. 4. Seq ID SJ201704 has been developed based upon rs4l284767SNP Sr No. 5. Seq ID SJ201705 has been developed based upon rsl800796SNP Sr No. 6. Seq ID SJ201706 has been developed based upon rsl800796SNP Sr No. 7. Seq ID SJ201707 has been developed based upon rsl787572SNP Sr No. 8. Seq ID SJ201708 has been developed based upon rsl787572SNP Sr No. 9. Seq ID SJ201709 has been developed based upon rsl0l2334SNP Sr No. 10. Seq ID SJ201710 has been developed based upon rsl0l2334SNP Sr No. 11. Seq ID SJ201711 has been developed based upon rsl2979860 SNP Sr No. 12. Seq ID SJ201712 has been developed based upon rsl2979860SNP Sr No. 13. Seq ID SJ201713 has been developed based upon rs22283l5SNP Sr No.l4. Seq ID SJ20l7l4has been developed based upon rs22283l5SNP Sr No. 15. Seq ID SJ201715 has been developed based upon rs28437lOSNP Sr No. 16. Seq ID SJ201716 has been developed based upon rs28437lOSNP Sr No. 17. Seq ID SJ201717 has been developed based upon rs763780SNP Sr No. 18. Seq ID SJ201718 has been developed based upon rs763780SNP Sr No. 19. Seq ID SJ201719 has been developed based upon rsl077467l SNP Sr No. 20. Seq ID SJ201720 has been developed based upon rsl077467l SNP Sr No. 21. Seq ID SJ201721 has been developed based upon rsl990760F SNP Sr No. 22. Seq ID SJ201722 has been developed based upon rsl990760SNP Sr No. 23. Seq ID SJ201723 has been developed based upon rs377529lSNP Sr No. 24. Seq ID SJ201724 has been developed based upon rs377529lSNP Sr No. 25. Seq IDrs SJ201725 has been developed based upon rsl 13181057 SNP Sr No. 26. Seq ID SJ20l726has been developed based upon rsl 13181057 SNP Sr No. 27. Seq ID SJ201727 has been developed based upon rsl08l383lSNP Sr No. 28. Seq ID SJ201728 has been developed based upon rsl08l383l SNP Sr No. 29. Seq ID SJ201729 has been developed based upon rs243056lSNP Sr No. 30. Seq ID SJ201730 has been developed based upon rs243056lSNP

[0021] Multiplex SNP Primers Sr No. 31. Seq ID SJ201731 has been developed based upon rsl800896SNP

Sr No. 32. Seq ID SJ201732 has been developed based upon rs4l284767SNP

Sr No. 33. Seq ID SJ201733 has been developed based upon rsl800796SNP

Sr No. 34. Seq ID SJ201734 has been developed based upon rsl787572SNP

Sr No. 35. Seq ID SJ201735 has been developed based upon rsl0l2334SNP

Sr No. 36. Seq ID SJ201736 has been developed based upon RS12979860SNP

Sr No. 37. Seq ID SJ201737 has been developed based upon rs22283l5SNP

Sr No. 38. Seq ID SJ201738 has been developed based upon rs28437lOSNP

Sr No. 39. Seq ID SJ201739 has been developed based upon rs763780SNP

Sr No. 40. Seq ID SJ201740 has been developed based upon rsl077467lSNP

Sr No. 41. Seq ID SJ201741 has been developed based upon rsl990760 SNP

Sr No. 42. Seq ID SJ201742 has been developed based upon rs377529lSNP

Sr No. 43. Seq ID SJ201743 has been developed based upon rsl l3l8l057SNP

Sr No.44. Seq ID SJ201744 has been developed based upon rsl08l383lSNP

Sr No. 45. Seq ID SJ201745 has been developed based upon rs243056lSNP

[0022] The present invention provides primers developed for detecting host genetic markers for Enterovirus A71 , wherein the primers comprise sequences selected from SEQ ID No. 1-45.

[0023] The primers comprising SEQ ID 1-30 are primers for Polymerase Chain Reaction (PCR).

[0024] The primers comprising SEQ ID 31-45 are primers for single nucleotide polymorphism (SNP) reaction.

[0025] The primers are used independently or multiplexed.

[0026] An assay for detecting host genetic risk markers for Enterovirus A71, wherein said assay comprises the steps of:

-obtaining deoxyribonucleic acid (DNA) from a sample;

-amplifying the obtained DNA using PCR technique;

-subjecting said amplified DNA to SNP reaction;

-identifying the SNPs for genotype of individual samples. [0027] PCR is performed using primers comprising sequences selected from SEQ ID NO. 1- 30.

[0028] SNP reaction is performed using primers comprising sequences selected from SEQ ID NO. 31-45. [0029] The assay is either performed with single set of primers or with a set of multiplex primers.

[0030] A method of diagnosing or identification of Enterovirus A71 related diseases, wherein said method comprises the steps of:

Isolating nucleotide from a sample; - conducting hybridization assay using the primers selected from SEQ ID No. 31-

45; quantifying the hybridized and non-hybridized nucleotides and accordingly comparing the same with an established standard to confirm the presence of the infection. [0031] The aforesaid nucleotide is selected from DNA or RNA.

[0032] The primers have radioactive or fluorescent tag.

[0033] A kit comprising primers as defined above.

[0034] DNA extracted from human blood using standard methods is subjected to multiplexed PCR followed by multiplexed SNP reactions using SNaPshot multiplexed kit (Applied Biosystems). Results are generated by capillary electrophoresis in Genetic Analyzer and Gene Mapper software.

BRIEF DESCRIPTION OF FIGURES:

The disclosure may be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows: [0035] Figure No 1A: Shows the results of Primer focus assay for7 SNPs. The 7 SNPs can be read without ambiguity.

[0036] Figure No IB: Shows the result of a human DNA samples. The 7 SNPs can be read without ambiguity. [0037] Figure No 2A: Shows the results of Primer focus assay for 8 SNPs. The 8 SNPs can be read without ambiguity.

[0038] Figure No 2B: Shows the result of a human DNA samples. The 8 SNPs can be read without ambiguity.

[0039] Figure No 3: Figure shows the SNP Multiplex results of a human DNA sample of Sample No 1. Set 1(7 SNPs can be read without ambiguity) and Set 2 (8 SNPs can be read without ambiguity). The experiments were conducted 3 times on same DNA sample to check the reproducibility of the SNP Multiplex results of 15 SNPs in 12 different genes.

[0040] Figure No 4: Figure shows the SNP Multiplex results of a human DNA sample of Sample No 2. Set 1(7 SNPs can be read without ambiguity) and Set 2 (8 SNPs can be read without ambiguity). The experiments were conducted 3 times on same DNA sample to check the reproducibility of the SNP Multiplex results of 15 SNPs in 12 different genes.

[0041] Figure No 5: Figure shows the SNP Multiplex results of a human DNA sample of Sample No 3. Set 1(7 SNPs can be read without ambiguity) and Set 2 (8 SNPs can be read without ambiguity). The experiments were conducted 3 times on same DNA sample to check the reproducibility of the SNP Multiplex results of 15 SNPs in 12 different genes.

[0042] Figure No 6: Figure shows the SNP Multiplex results of a human DNA sample of Sample No 4. Set 1(7 SNPs can be read without ambiguity) and Set 2 (8 SNPs can be read without ambiguity). The experiments were conducted 3 times on same DNA sample to check the reproducibility of the SNP Multiplex results of 15 SNPs in 12 different genes.

[0043] Figure No 7: Figure shows the SNP Multiplex results of a human DNA sample of Sample No 5. Set 1(7 SNPs can be read without ambiguity) and Set 2 (8 SNPs can be read without ambiguity). The experiments were conducted 3 times on same DNA sample to check the reproducibility of the SNP Multiplex results of 15 SNPs in 12 different genes. [0044] Figure no 8. The results of 5 samples of SNP (RS1800896) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0045] Figure no 9. The results of 5 samples of SNP (RS41284767) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0046] Figure no 10. The results of 5 samples of SNP (RS1800796) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0047] Figure no 11. The results of 5 samples of SNP (RS1990760) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0048] Figure no 12. The results of 5 samples of SNP (RS1012334) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram. [0049] Figure no 13. The results of 5 samples of SNP (RS12979860) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0050] Figure no 14. The results of 5 samples of SNP (RS2228315) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram. [0051] Figure no 15. The results of 5 samples of SNP (RS2843710) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0052] Figure no 16. The results of 5 samples of SNP (RS763780) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram. [0053] Figure no 17. The results of 5 samples of SNP (RS10774671) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0054] Figure no 18. The results of 5 samples of SNP (RS1787572) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0055] Figure no 19. The results of 5 samples of SNP (RS3775291) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0056] Figure no 20. The results of 5 samples of SNP (RS113181057) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0057] Figure no 21. The results of 5 samples of SNP (RS10813831) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

[0058] Figure no 22. The results of 5 samples of SNP (RS2430561) peak was confirmed by Sanger Sequencing Method. SNPs were marked in the chromatogram.

DETAILED DESCRIPTION OF THE INVENTION

[0059] At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.

[0060] Throughout the description, the phrases“comprise” and“contain” and variations of them mean“including but not limited to”, and are not intended to exclude other moieties, additives, components, integers or steps. Thus, the singular encompasses the plural unless the context otherwise requires. Wherever there is an indefinite article used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. [0061] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification including any accompanying claims, abstract and drawings or any parts thereof, or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0062] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Post filing patents, original peer reviewed research paper shall be published.

[0063] The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. [0064] Unless contraindicated or noted otherwise, throughout this specification, the terms“a” and“an” mean one or more, and the term“or” means and/or.

The present invention includes designing of 15 SNP primers along with corresponding 30 new PCR/ sequencing primers for detection of Single Nucleotide Polymorphism (SNP) in 12 different genes. The SNPs are involved in genetic susceptibility, disease progression, and effectiveness of treatment for EV-A71 infection. The SNP reactions of RS 1800896 and RS2228315 has been done in the reverse direction. The SNP alleles for these SNPs are reported to be in reverse orientation with respect to the Genome. (As per NCBI, dbSNP report).

[0065] The PCR primers and SNP primers have been so arranged to develop 2 new multiplex SNP detections assays, one for 7 SNPs and the others for 8 SNPs. The assay design confirms that the SNP peaks appear distinct with no overlap or ambiguity in interpretation of the results. Taken together the novel assay provides genotype information of 15 SNPs for the test samples. Details of invention are given in below:

[0066] The inventors have utilized 15 single nucleotide polymorphisms in 12 genes as target for developing the assay of the present invention. The inventors have artificially designed new primers for PCR amplification of specific gene segment flanking the SNPs in forward and reverse direction. Using the forward and reverse PCR primer the specific gene segments were amplified. The correctness of the amplification was confirmed by sequencing using the same primers and BLAST search. The inventor artificially designed novel SNP primers of different lengths and overhangs that are one nucleotide short of the targeted SNPs. The details of the primers and genes/ SNPs targeted are given in Table 2.

Table 2: details of PCR Primers and SNP detection Primers included in the claim:

[0067] SNP primers are of different lengths with non-complementary overhangs. The purpose of the over hangs was to provide different electrophoresis migration during capillary electrophoresis. SNP primers were checked individually to confirm correct identification of the SNP sites using SNapShot Multiplex reagent (Applied Biosystems, Foster City, CA). The inventors checked the SNP primers by primer focus assay (Applied Biosystems, Foster City, CA) to determine migration pattern of all 4 nucleotide incorporation in Capillary Electrophoresis. Using the primer focus data the SNP reaction were multiplex into 2 separate assay containing 7 and 8 SNP reaction respectively. The PCR primers were multiplexed as per the SNP primer multiplexing. The details of the 2 SNP assays (7X and 8X multiplexed) are given in (Table 3).

Table 3: Details of new and novel primers for PCR and SNP multiplex assay as per targeted SNP

Final outline of the Assay

[0068] Source of the test materials: Human DNA extracted from any type of cells can be used in the present assay. The most preferred samples may be 2-3 drops of blood from finger tips collected into an EDT A/Heparin contacting test tube.

Steps involved for the assay:

1. DNA samples from clinical subjects.

2. PCR amplification using 7 multiplexed PCR and 8 multiplexed PCR by Phusion Blood Direct PCR Kit (FINZYMES, Thermo Scientific, USA).

3. PCR product purification using QIAquick Gel Extraction Kit (QIAGEN, Germany) as per the manufacturer’s instructions.

4. 7X multiplexed and 8X multiplexed reactions using SNapShot Multiplex kit (Thermo Scientific, USA) as per manufacturer protocol.

5. Capillary Electrophoresis in Genetic Analyser (Applied Biosystems, Foster City, CA)

6. Data retrieval using Gene Mapper Software (Applied Biosystems, Foster City, CA).

7. Tabulation of the 15 SNP results for the genotype of individual samples.

8. The assay is suitable for determining the genetic susceptibility markers of EV71 in patients as well as populations. [0069] To the best of our knowledge there is no similar or equivalent assay available as of date.

[0070] Now, the protocol followed is deliberated through example, the scope of which may vary as per the dynamic factors taken into consideration.

Example 1:

[0071] Study design: Random samples survey for proportion/ prevalence (Diagnostics and epidemiology)

Primer Focus reaction (Applied Bio systems): [0072] Primer focus reaction adds a single molecule of each of the 4 nucleotides to the

SNP primer. The reaction is not template dependent. A single molecule of A/T/G/C added to the SNP primer can cause small but significant change in the electrophoretic mobility. Thus, though the length of the SNP primer increases by 1 nucleotide only migration after A/T/G/C addition may be different. In multiplexed SNP reactions care is necessary to ensure that adjacent SNP reactions do not overlap even after addition of any of the 4 nucleotides for confident reading.

Example 2:

[0073] Multiplex Assay 1: 7X-multiplexed SNP assay

[0074] 7X-multiplexed SNP detection assay to determine genotype of 7 different SNPs in a human DNA sample.

[0075] Human DNA sample was subjected to seven individual PCR for the indicated genes/ SNPs. Amplification products were purified by PCR purification kit. Primer Focus reactions were performed for the 7 PCR products using specific SNP Primer individually. Equal volumes of Primer Focus reaction of the 7 SNP primers were mixed and electrophoresed in a Genetic Analyzer. The electrophoretic separation was analyzed by GeneMapper Software. The SNP primers were artificially designed of different lengths for their clear separation after capillary electrophoresis. The smallest length primer appears to the left and the larger length primers appear to the right in order of increase length. Primer focus reaction (Test) confirmed that the different SNP reactions remain distinct even after addition of a single molecule of any of the four nucleotides. Figure shows that each SNP reaction was clearly separate from the other so that there would be no ambiguity in determining the nucleotides present as SNP. Gene name and SNP ID are shown close to the coloured peaks. Peaks are colour coded as Green = Adenine (A), Black = Cytosine (C), Blue = Guanine (G) and Red = Thymidine (T). The result is depicted vide Figure 1A.

[0076] 7X-multiplexed SNP assay performed on a human DNA sample. DNA sample was subjected to 7X multiplexed PCR reaction. The PCR amplification product was purified through PCR purification kit (QIAGEN). 7X-multiplexed SNP assay was carried out using SNapShot kit (Thermo Scientific, USA) and electrophoresed in a Genetic Analyzer 3130XL (Applied Biosystems, USA). Data was retrieved using GeneMapper software (Applied Biosystems, USA). The results are depicted in Figure 1B.

[0077] The 7 SNPs can be identified as (from left to right)

1. ILl0/ rsl800896=G

2. SCARB2/ rs4l284767=T/C

3. IL6/ rsl800796=G

4. MDA-5/rs 1990760 =T

5. IFNARl_Intron3/ rsl0l2334= ATT

6. IFNL-4/ rsl2979860= T/C

7. SELPLG Exon/ rs22283l5=C

Example 3:

[0078] Multiplex Assay 2: 8X-multiplexed SNP assay

[0079] 8X-multiplexed SNP detection assay to determine genotype of 8 different SNPs in a human DNA sample.

[0080] Fluman DNA sample was subjected to seven individual PCR for the indicated genes/ SNPs. Amplification products were purified by PCR purification kit. Primer Focus reactions were performed for the 8 PCR products using specific SNP Primer individually. Equal volumes of Primer Focus reaction of the 8 SNP primers were mixed and electrophoresed in a Genetic Analyzer. The electrophoretic separation was analyzed by GeneMapper Software. The SNP primers were artificially designed of different lengths for their clear separation after capillary electrophoresis. The smallest length primer appears to the left and the larger length primers appear to the right in order of increase length. Primer focus reaction (Test) confirmed that the different SNP reactions remain distinct even after addition of a single molecule of any of the four nucleotides. Figure shows that each SNP reaction was clearly separate from the other so that there would be no ambiguity in determining the nucleotides present. Gene name and SNP ID are shown close to the coloured peaks. Peaks are color coded as Green = Adenine (A), Black = Cytosine (C), Blue = Guanine (G) and Red = Thymidine (T). This has been depicted through Figure 2A. [0081] 8X-multiplexed SNP assay performed on a human DNA sample. DNA sample was subjected to 8X multiplexed PCR reaction. The PCR amplification product was purified through PCR purification kit (make). 8X-multiplexed SNP assay was carried out using SNapShot (kit from Thermo Scientific, USA) and electrophoresed in a Genetic Analyzer 3130XF (Applied Biosystems). Data was retrieved using GeneMapper software. This has been represented by Figure 2B.

[0082] The 8 SNPs can be identified as (from left to right)

1. IFNAR1/ rs28437lO=G/C

2. Interleukin l7/rs763780 =T

3. O AS l/rsl077467l=A

4. IFNAR1/ rsl787572=G

5. TFR-3/rs3775291 =C/T

6. IFNARl/rsl l3l8l057=T

7. RIGT/rsl08l383l=G/A

8. IFNy-RS2430561 = A/T

[0083] Example 3: Standardization of the assay.

5 samples were collected, and assay of the present invention was performed to test a) reproducibility and specificity (b). The results of 15 SNPs of the 5 samples were confirmed by sequencing the PCR amplification products to prove that the correct gene segments are amplified, and nucleotide position confirmed. The SNP reactions of RS 1800896 and RS2228315 has been done in the reverse direction, hence as shown in the below table 4, the SNP alleles for these SNPs are reported to be in reverse orientation with respect to the Genome.

The results are provided in Figures 3- 22 and Table 4 and Table 5 below: Table 4:

Setl: Multiplex SNP results were confirmed by Sequencing results.

RS1800896 and RS2228315: SNP alleles are reported in Revers Orientation with respect to the Genome (As per NCBI, dbSNP report).

Table 5:

Set2: Multiplex SNP results were confirmed by Sequencing results.