FIELD OF INVENTION

The described invention is in the field of diagnosis and characterization of virus specifically, application of single base pair extension technique to identify and differentiate Feline Panleukopenia Virus and/or multiple types of Canine Parvovirus in a single reaction using the oligonucleotides as disclosed in the present invention.

BACKGROUND OF THE INVENTION

In 1978 a new parvovirus, very closely related to FPV, was first described in dogs (Carmichael, 2005). It was named canine parvovirus type 2 (CPV-2), to distinguish it from another parvovirus isolated from dogs in 1970, which is now called canine minute virus (CaMV). Feline panleukopenia virus (FPV) is an autonomous parvovirus. It is the prototype of a number of closely related parvoviruses which were isolated from various carnivores such as dogs, mink, raccoons, raccoon dogs, foxes and other canids (Parrish, C. R. 1990. Emergence, natural history, and variation of canine, mink and feline parvoviruses. Adv. Virus Res. 38:403-450). The viruses were initially named after the hosts from which they had been isolated. Current taxonomy defines canine parvovirus and feline panleukopenia virus as one single taxonomic entity (Tattersall, 2006), but in the present guidelines, FPV refers to parvovirus in cats. FPV is known to infect cats and other members of the Felidae, as well as raccoons, mink, and foxes (Steinel et al., 2001). In dogs, FPV replication was seen only in lymphoid tissues, such as thymus, spleen and bone marrow, but not in the gut, and the virus is not shed (Truyen and Parrish, 1992).

CPV-2 is believed to have evolved from FPV by acquiring 5 or 6 amino acid changes in the capsid protein gene (Truyen, 1999). Interestingly, CPV-2 was no longer able to infect cats. However, during subsequent further adaptation to the canine host, the amino acid changes that had enabled the new virus to better bind to the canine cellular receptor also resulted in its ability to infect cats (Hueffer and Parrish, 2003). The parvoviruses now circulating in the dog populations worldwide and which can be genetically and antigenically defined as the types CPV-2a, -2b, and "-2c", are able to infect cats and may even cause disease (Truyen et al., 1995, 1996; Mochizuki et al., 1996). However, feline CPV infections are rare in Europe and the USA, and CPV is found only sporadically in diagnostic material (Truyen et al., 1996). CPV was isolated from feline peripheral blood lymphocytes after numerous blind passages, and viral DNA was demonstrated by polymerase chain-reaction (PCR), as reported in a study from Taiwan (Ikeda et al., 2000).

Canine parvovirus (CPV) is a single stranded DNA virus that causes acute and sometimes fatal enteritis in dogs. The virus was identified in dogs during 1977-1978 as CPV type 2 (Appel, M.J., Scott, F.W., Carmichael, L.E., 1979. Isolation and immunisation studies of a canine parvo-like virus from dogs with haemorrhagic enteritis. Vet. Rec. 105, 156-9; Burtonboy, G., Coignoul, F., Delferriere, N., Pastoret, P.P., 1979. Canine hemorrhagic enteritis: detection of viral particles by electron microscopy. Arch. Virol. 61, 1-11.). Soon after the identification of the CPV2, an antigenic variant, CPV2a, started appearing in the canine population (Parrish, C.R., O'Connell, P.H., Evermann, J.F., Carmichael, L.E., 1985. Natural variation of canine parvovirus. Science. 230, 1046-8.). In the mid 1980s, another CPV2 variant called CPV2b appeared (Parrish, C.R., Aquadro, C.F., Strassheim, M.L., Evermann, J.F., Sgro, J.Y., Mohammed, H.O., 1991. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J. Virol. 65, 6544-6552.). The CPV type 2 was replaced completely by the variant types 2a and 2b all over the world and these two variants co-exists in the canine population in varying proportions (Truyen, U., Evermann, J.F., Vieler, E., Parrish, C.R., 1996. Evolution of canine parvovirus involved loss and gain of feline host range. Virology 215, 186-189; Chinchkar, S.R., Mohana Subramanian, B., Hanumantha Rao, N., Rangarajan, P.N., Thiagarajan, D., Srinivasan, V.A., 2006. Analysis of VP2 gene sequences of canine parvovirus isolates in India. Arch. Virol. 151, 1881-1887; Pereira, C.A.D., Leal, E.S., Durigon, E.L., 2007. Selective regimen shift and demographic growth increase associated with the emergence of high fitness variants of canine parvovirus. Infect. Genet. Evol. 3, 399- 409). The characteristic amino acid changes in the capsid protein (VP2), which changes the CPV type from 2 to 2a and to 2b, are very limited. Substitutions at positions 87 (Met to Leu), 300 (Gly to Ala), 305 (Tyr to Asp) and 555 (Val to lie) are responsible in the evolution of 2 to 2a. Similarly, substitutions at positions 426 (Asn to Asp) and 555 (IIe to Val) are involved in the emergence of 2b from 2a (Truyen,U.,Gruenberg,A.,Chang, S.F.,Obermaier, B.,Veijalainen, P., Parrish, C.R., 1995. Evolution of the feline subgroup parvoviruses and the control of canine host range in-vivo. J.Virol.69, 4702—4710). However, it was reported that many of the recent 2a strains do not carry the Val to IIe substitution at position 555 (Wang, H.C., Chen,W.D., Lin, S.L., Chan, J.P.W.,Wong, M.L., 2005. Phylogenetic analysis of canine parvovirus VP2 gene in Taiwan. Virus Genes 31, 171-174; Martella, V., Decaro, N., Buonavoglia, C., 2006. Genetic and antigenic variation of CPV-2 and implicance in antigenic/genetic characterization. Virus Genes 33, 11-13; Chinchkar et al., 2006). Thus, a single amino acid change can differentiate the VP2 sequences of CPV2a and CPV2b (at position 426). Another CPV variant (CPV 2c) having glutamic acid at position 426 was reported from various parts of the world (Buonavoglia, C., Martella, V., Pratelli, A., Tempesta, M., Cavalli, A., Buonavoglia, D., Bozzo, G., Elia, G., Decaro, N., Carmichael, L.E., 2001. Evidence for evolution of canine parvovirus type-2 in Italy. J. Gen. Virol. 82, 3021-3025; Nakamura, M., Tohya, Y., Miyazawa, T., Mochizuki, M., Phung, H.T.T., Nguyen, N.H., Huynh, L.M., Nguyen, L.T., Nguyen, P.N., Nguyen, P.V., Nguyen, N.P., Akashi, H., 2004. A novel antigenic variant of canine parvovirus from a Vietnamese dog. Arch. Virol. 149, 2261-2269).

Taken together, these mutations restrict the differences among the antigenic variants of CPV2 to only one amino acid at position 426 (i.e. Asn in CPV-2a, Asp in CPV-2b and Glu in CPV-2c) and in turn only one nucleotide change at positions 4042 or 4064. The evolution observed in the VP2 of CPV-2 has important consequences for the PCR-based genotyping assay described by Pereira et al (Pereira, C. A., T.A. Monezi, D.U. Mehnert, M. D'Angelo, E.L. Durigon, Vet. Microbiol. 75,127-133; 2000).

Due to the limited number of nucleotide variations between CPV-2a and -2b, the 2b- specific primers were earlier selected taking advantage of two single nucleotide polymorphisms (SNPs), 4062-A T and 4449-G → A, that determine the replacement of Asn by Asp at position 426 and of lie by Val at position 555, in reference CPV-2a and -2b strains, respectively. Each primer was selected to have one such mutation at the very 3' end, as nucleotide mismatches that occur at the 3' end of a primer are highly detrimental to primer extension and strongly decrease PCR amplification. However, most new CPV-2a strains have the mutation 555 Val, due to the nucleotide mutation 4449- G→ A. Therefore, the PCR-based genotyping system developed by Pereira et al. (2000) is no longer able to discriminate between type-2a and type-2b strains, as almost all the novel CPV-2a strains (555-Val) will go mischaracterized as type-2b. In addition, the genotyping system is not able to identify the Glu-426 antigenic variant. The nucleotide change responsible for the mutation Glu-426 has occurred 2 nucleotides downstream the primer binding region and thus cannot be detected in this PCR-based genotyping system. To address this point, it is necessary to adopt molecular assays able to recognize SNPs. Restriction enzyme digestion of short PCR fragments of CPV-2 genome proved to be useful to discriminate between CPV-2c and other variant types (2b and 2a) (Buonavoglia et al., 2001). The type 2c CPV strains harbors an MboII cleavage site at 4064 that could be detected in PCR RFLP (restriction fragment length polymorphism). However, the PCR-RFLP can only differentiate CPV 2c from other variants and the differentiation in between the 2a and 2b cannot be achieved. Moreover, Demeter et al. (Demeter, Z., Palade, E.A., Soo's, T., Farsang, A., Jakab, C., Rusvai, M., 2010. Misleading results of the MboII-based identification of type 2a canine parvovirus strains from Hungary reacting as type 2c strains. Virus Genes. 41, 37-42) reported false positive results using the PCR-RFLP method in identifying CPV type 2c.

Apart from this, two minor groove binder (MGB) assays using real time PCR was also developed to recognize the SNPs observed in the variants of CPV-2 (Decaro, N., Gabriella Elia, Vito Martella, Marco Campolo, Costantina Desario, Michele Camero, Francesco Cirone, Eleonora Lorusso, Maria Stella Lucente, Donato Narcisi, Pierluigi Scalia, Canio Buonavoglia. 2006. Characterisation of the canine parvovirus type 2 variants using minor groove binder probe technology. Journal of Virological Methods 133: 92-99). Using this technique, several individual assays had to be performed at each SNP site to differentiate the types 2a, 2b and 2c from a sample. Moreover, the presence of CPV type 2 and 2b vaccine strains in the field further add up to the complication. Newer mutations (Battilani, M., Balboni, A., Ustulin, M., Giunti, M., Scagliarini, A., Prosperi, S., 2011. Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats. Vet. Res. 42, 43) at the SNP sites can not be detected using the MGB assays without changing any of the assay protocol or reagents. Additional probes will have to be designed and incorporated based on the changes in the SNP sites.

Single nucleotide extension using the mini-sequencing technique is one of the methods used to identify SNPs. The primers were designed in such a way that the 3' end of the primer is one base short of the known SNP. In the mini-sequencing reaction, primers bind to a complementary template in the presence of fluorescently labeled ddNTPs and the polymerase extends the primer by one nucleotide. All the four ddNTPs were labeled with four different dyes. The SNP detection is then possible by separating extended products and detecting the fluorescence by capillary gel electrophoresis. Thus, the single nucleotide polymorphisms (SNPs) at known locations can be identified. Various multiplex minisequencing-based assays have been successfully validated for the analysis of mtochondrial DNA, autosomes, the Y-chromosome, Duffy and ABO group system, and the melanocortin 1 receptor gene. Thus, the technique was used in genotyping human genomes by SNPs. The mini-sequencing approach using single base extension was so far not reported for typing or characterizing any virus genome.

SUMMARY OF THE INVENTION

One of the aspect of the present invention provides a set of oligonucleotides for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the set comprises oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO:13.

Another aspect of the present invention provides a kit for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the kit comprises a set of oligonucleotides comprising oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 13, dNTPs, ddNTPs, polymerase and reaction buffer.

Yet another aspect of the present invention provides a process for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the process comprises providing a sample potentially containing at last one target nucleic acid sequence; performing a polymerase chain reaction using a reaction mixture comprising the sample and the oligonucleotides as set forth in SEQ ID NO: 6 and SEQ ID NO: 7 to obtain an amplified product; performing a mini-sequencing reaction using a mini-sequencing reaction mixture comprising the amplified product, and the oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 13 to obtain mini-sequencing product; separating the mini-sequencing products by size or electrophoretic mobility; and detecting the presence of the target nucleic acid sequence by distinguishing the separated mini- sequencing product, wherein the target nucleic acid sequence is of FPV, CPV2, CPV2a, CPV2b or CPV2c.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 shows the mini-sequencing peaks obtained for various CPV types and FPV after capillary gel electrophoresis. The mini-sequencing reaction mix was run in ABI PRISM 3130x1 genetic analyzer. The data were collected using 3130x1 version 3 software and analyzed using the Gene-mapper v.3.7 software (Applied Biosystems, USA). The SNP position in the CPV genome is provided on the top over the corresponding peak. Mixed template of type 2a and 2b produced double peak at the relevant position (4062). A = green; C = black; G = blue; T = red.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

The terms "polynucleotide", "nucleotide", "DNA", "gene" and "nucleic acid" are used interchangeably.
The terms "primer" and "oligonucleotide" are used interchangeably.

In the present invention, the "target nucleic acid sequence" embraces not only a nucleic acid sequence to be amplified but also a sequence complementary thereto.

The present invention provides oligonucleotides having nucleotide sequence as set forth in SEQ ID NO: 1 to 7 and SEQ ID NO:13. The present invention also provides a process for detection and differentiation of FPV (Feline Panleukopaenia Virus), CPV2, CPV2a, CPV2b and CPV2c in a single reaction using mini-sequencing technique. Using this technique multiple CPV types can be identified in a single reaction and the mixed infections can also be identified.

The primers as disclosed in the present invention encompass labeled primers also.

Currently, the confirmation of the types (in particular type 2a and 2b) is done by sequencing of the genome. Thus, the identification of five types of virus in a single reaction is a technical advancement in the field of virus typing. Moreover, the process as disclosed in the present invention also has the scope to identify multiple types of viruses present in the mixed infections. Therefore, it can be used (1) to identify the types of CPV which are co-infecting dogs (2) identify the CPV vaccine virus types co- circulating in the population along with the virulent virus types. The process has the scope to include five more primers (and a maximum often primers); thereby emerging virus types and vaccine strains can also be identified. Diagnostic applicability for this method is (1) use of the kit to type the Parvovirus rapidly and reliably (2) Use of the kit to differentiate vaccine and field viral strains. (3) This technique can be used as a tool in laboratories for Parvovirus characterization.

The nucleotides sequence as set forth in SEQ ID NO: 8 to 11 shows four different sequences representing CPV2a, CPV2b, CPV2, and CPV2c. The FPV nucleotides sequence is as set forth in SEQ ID NO: 12.

The oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 13 of the present invention are capable of identifying all four types of CPV viz. CPV2a, CPV2b, CPV2, and CPV2c ; and FPV.

The present invention is elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention.

In accordance with the present invention in one of the embodiment, there is provided a set of oligonucleotides for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the set comprises oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 13.

In another embodiment of the present invention there is provided a set of oligonucleotides for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the set comprises oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 13, wherein the Canine Parvovirus (CPV) type is selected from a group consisting of CPV2, CPV2a, CPV2b and CPV2c.

Another embodiment of the present invention provides a kit for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the kit comprises the set of oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 13, dNTPs, ddNTPs, polymerase and reaction buffer.

Another embodiment of the present invention provides a kit for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the kit comprises the set of oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 13, dNTPs, ddNTPs, polymerase and reaction buffer.

Yet another embodiment of the present invention provides a process for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the process comprises providing a sample potentially containing at last one target nucleic acid sequence; performing a polymerase chain reaction using a reaction mixture comprising the sample and the oligonucleotides as set forth in SEQ ID NO: 6 and SEQ ID NO: 7 to obtain an amplified product; performing a mini-sequencing reaction using a mini-sequencing reaction mixture comprising the amplified product, and the oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 13 to obtain mini-sequencing product; separating the mini-sequencing products by size or electrophoretic mobility; and detecting the presence of the target nucleic acid sequence by distinguishing the separated mini- sequencing product, wherein the target nucleic acid sequence is of FPV, CPV2, CPV2a, CPV2b or CPV2c.

Yet another embodiment of the present invention provides a process for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the process comprises providing a sample potentially containing at last one target nucleic acid sequence; performing a polymerase chain reaction using a reaction mixture comprising the sample and the oligonucleotides as set forth in SEQ ID NO: 6 and SEQ ID NO: 7 to obtain an amplified product of 576 bp; performing a mini-sequencing reaction using a mini-sequencing reaction mixture comprising the amplified product, and the oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 13 to obtain mini-sequencing product; separating the mini-sequencing products by size or electrophoretic mobility; and detecting the presence of the target nucleic acid sequence by distinguishing the separated mini- sequencing product, wherein the target nucleic acid sequence is of FPV, CPV2, CPV2a, CPV2b or CPV2c.

Identifying more than one CPV types simultaneously from mixed infection

The mini-sequencing reaction was performed for the DNA extracted from the cell culture supernatant of mixed infection of two different CPV types. Similarly, the typing efficiency in mixed infection was evaluated by mixing varying copy numbers of the CPV plasmid constructs. Mixed infections were identified by the presence of double peaks in the pertinent SNP positions (Figure 1). The sensitivity of detection did not change significantly in identifying the individual CPV types though the typing was done in the presence of more than one CPV type. Natural infection of dogs with more than one CPV types was reported earlier (Battilani et al., 2011; Vieira, M.J., Silva, E., Desario, C., Decaro, N., Carvalheira, J., Buonavoglia, C., Thompson, G., 2008. Natural Co-infection with 2 Parvovirus Variants in Dog. Emerg. Infect. Diseases. 14, 678-679). The mini-sequencing method described in this manuscript can detect mixed infection in dogs with more than one CPV types in a single reaction. As the mini- sequencing technique can accommodate up to ten primers, there is scope for adding few more primers in the reaction to identify any further mutations originating from other sites of VP2 gene. The SNPs present in any site outside the 576 bp region used in the present study can also be detected by performing multiplex PCR to amplify the relevant regions. Thus, emerging SNP sites and type 2b vaccine strain specific SNPs can also be added in future.

EXAMPLES

Examples 1

Polymerase Chain Reaction to amplify a part of VP2

Canine Parvovirus isolates from our repository was passaged in A72 cell line. Total DNA was extracted from the virus infected tissue culture supernatant using DNaZol reagent. The PCR reaction mixture contained 400 μM each of dNTPs, 1 unit Taq Polymerase, 1.5 mM MgCl2, 3 pico moles each of primers and lOOng of DNA. The PCR reaction was performed in a total volume of 25 μl. The thermal cycling was done with an initial denaturation step of 94°C for 3 minutes; 40 cycles of 94°C for 45 seconds, 50°C for 45 seconds and 72°C for 1 minute; and final extension of 72°C for 15 minutes. The primer pair, SEQ ID NO: 6 and SEQ ID NO: 7, was used to amplify a part of VP2 gene from Canine Parvoviral DNA (Table 1). These primers were selected from the conserved regions in VP2 (DQ182612 to DQ182627) and had 100% homology with the VP2 nucleotide sequences of all the CPV types and FPV. Additionally, the region of CPV VP2 amplified by the PCR encompasses the SNPs which were sufficient to differentiate various CPV types. The amplified fragments were 576bp in length and contained the sequences in between 3622bp and 4197bp of the CPV genome (The numbering is based on the full length CPV sequence; GenBank accession number M38245).

Examples 2

Development of standard plasmid constructs

The PCR amplified partial VP2 coding sequences of CPV type 2, 2a and 2b were cloned into Topo TA cloning vector (Invitrogen, USA) separately and used as positive standard construct. Similar standard constructs for CPV type 2c and FPV were prepared by creating point mutations in CPV type 2b and CPV type 2 constructs, respectively. For creating CPV 2c construct, VP2 region of CPV 2b was amplified as two fragments with 20 bases overlap between the amplified fragments. The primers used to create the overlap contained the mutation and the mutation is incorporated in the amplified fragments (T-»A at 4064). The two fragments were subjected to splicing by overlap extension (SOE) PCR to create single CPV 2c amplicon. Then the mutated PCR product was cloned into Topo TA cloning vector (Invitrogen, USA). Similarly, the FPV construct was created from CPV2 plasmid construct by incorporating A-»G mutation at 3753 using SOE PCR. All the plasmid constructs were verified by sequencing.

Examples 3

PCR product treatment using Exonuclease I and Alkaline Phosphatase

The 576bp PCR product (obtained from viral DNA and plasmid clones) was added with 5 units of Shrimp Alkaline Phosphatase (SAP; New England Biolabs, USA) and 3 units of Exonuclease I (Exo I; New England Biolabs, USA). The mixture was incubated at 37°C for 1 hour to remove the unused primers and to dephosphorylate the unincorporated dNTPs. After the treatment, SAP and Exo I were inactivated by heating at 75°C for 15 minutes. The Exo/SAP treated PCR product was used directly as template in the subsequent mini-sequencing reaction.

Examples 4

Determining the relative mobility of labeled primers

Six primers (SEQ ID NO: 1 to 5 and SEQ ID NO:13; Table 1) were used in the multiplex mini-sequencing reaction. The mini-sequencing primers were designed in such a way that the 3' end of the primer is short by one base binding at nucleotide positions 3685, 3699, 3753, 4062 and 4064 in CPV genome (Table 1). The type- specific amino acid and the corresponding SNPs at these positions are shown in table

1. The mini-sequencing primers were 5' tailed with a non-homologous sequence dGACT to produce single base extension products ranging from 25 to 55 nucleotides. Thus, these extension products differ in length from each other by at least 7 nucleotides, allowing them to be sufficiently resolved in capillary electrophoresis. When the primers were extended using a labeled ddNTP in the mini-sequencing reaction, the presence of dye in the added ddNTP changes the relative mobility of the primers considerably. Thus, the expected relative mobility of the primers in the capillary gel was identified by adding single ddNTP to each of the primer used in the multiplex reaction. The addition of the labeled ddNTPs was done using primer focus kit (Applied Biosystems, USA) and the length of the primers with the fluorescently labeled ddNTP was identified in genetic analyzer. The expected relative mobilities of the primers containing an additional labeled ddNTP are provided in table 2. The mobility shift was more (9 to 12 bases) for shorter primers and the shift was lesser (5 to 7 bases) for longer primers. Three primers (SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5) out of six primers (SEQ ID NO: 1 to 5 and SEQ ID NO: 13) used in the mini-sequencing reaction were anti-sense primers.

Examples 5

Multiplex mini-sequencing reaction

A multiplex reaction was designed to identify FPV, CPV2, CPV2a, CPV2b and CPV2c in a single tube. The multiplex reaction was performed in a 10μl total volume containing 3 μl of Exo I/SAP treated PCR product, 10 picomoles each of the primers (SEQ ID NO: 1 to 5 and SEQ ID NO:13; Table 1) and 5μl SNaPshot™ multiplex ready reaction mix (Applied Biosystems, USA). SNaPshot™ reaction mix contained AmpliTaq® DNA polymerase, reaction buffer and fluorescently labeled ddNTPs (A = dR6G, green; C = dTAMRA™, black; G = dR110, blue; T = dROX™, red). The reaction mix was subjected to 28 cycles of 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 30 seconds. During the thermal cycling all the 6 primers get extended by single ddNTP. After extension, the mix was added with SAP (1 unit) and incubated at
37°C for 1 hour to remove the 5' phosphoryl groups of the unincorporated labeled ddNTPs. Then the SAP inactivation was done by heating at 75°C for 15 minutes. 0.5 |o.l of SAP treated extension product was mixed with 9 }il of formamide and 0.5 (il of internal size standard GeneScan-120 LIZ™ (Applied Biosystems, USA). The mix was heated at 95°C for 5 minutes and snap chilled on ice to denature the product. After denaturation, the samples were run on an ABI PRISM 3130x1 Genetic Analyzer using POP-7 polymer (Applied Biosystems, USA) in 36 cm capillary. Data were collected using 3130x1 (version 3) software and analyzed using the Gene-mapper v.3.7 software (Applied Biosystems, USA). Relative fluorescence intensity (RFU) of more than 100 was considered as positive peak.

The typical mini-sequencing signatures based on the type specific SNPs are provided in the table 2. These five SNPs were identified in the mini-sequencing reaction by the addition of specific ddNTP to the primers. Thus, the CPV types were identified without any ambiguity in the mini-sequencing reaction (figure 1). Mini-sequencing pattern typical for each of these viruses (or plasmid constructs) was obtained in the experiment.

Examples 6

Sensitivity of CPV typing using the mini-sequencing reaction

The Plasmid constructs containing part of VP2 sequences of CPV 2, 2a, 2b, 2c and FPV were 10 fold serially diluted starting with 107 copy numbers. 100 ng of DNA extracted from A72 cells was also mixed with each dilution of plasmid DNA. The PCR and subsequent mini-sequencing reaction was performed for each dilution of plasmid. The sensitivity was determined by finding out the lowest copy number of plasmid which produced unambiguous (>100 RFU) typing result. Each of the triplicate reactions were repeated thrice and the mean sensitivity was calculated. The distinct CPV typing could be arrived with less than 100 copies of the plasmids per reaction. The mean sensitivity was in between 18 to 60 plasmid copies per reaction for different CPV types (table 3). Detection limits of the type-specific MGB probe real time-PCR assays were 10' and 10 DNA copies for types 2a and 2b, respectively (type 2a/2b assay), and 102 and 101 DNA copies for types 2b and 2c, respectively (type 2b/2c assay; Decaro et al., 2006). Thus, the detection limit of mini-sequencing reaction is comparable with that of MGB probe assay. Moreover, the typing by mini-sequencing could be achieved in a single tube. As the mini-sequencing specifically incorporates a single complimentary base from the four possible ddNTPs, newer mutations (Battilani et al., 2011) at the SNP sites could also be detected without changing any of the assay protocol or reagents. However, in the MGB assay additional probes will have to be designed and incorporated based on the changes in the SNP sites.

CPV type 2, 2a and 2b virus were titrated in A72 cells using fluorescently labeled CPV specific mAb following the procedure described by Becerra and Hegland (Becerra, V.M., Hegland, M.J., 2005. Supplemental Assay Method for the Titration of Canine Parvovirus in Cell Culture. In: United States Department of Agriculture Center for Veterinary Biologies Testing Protocol. APHIS manual) with few modifications. The titrated viruses were 10 fold serially diluted from 105 FAID50 and each of these dilutions of viruses was spiked in faeces collected from CPV free dogs. Sensitivity of the mini-sequencing reaction was estimated using DNA extracted from the faeces samples containing different dilutions of virus. The DNA was PCR amplified and subjected to mini-sequencing reaction. The lowest dilution of virus required to obtain an explicit (>100 RFU) typing result by mini-sequencing was determined. The sensitivity experiment was done in triplicates and repeated on three different days. The mean sensitivity in terms of FAID50 was calculated. Cycle sequencing reaction was also performed using the PCR products to determine the lowest amount of virus titer required to get unambiguous sequencing reads. The CPV typing by mini-sequencing was recognizable distinctly even with the DNA extracted from <10* FAID50 of virus spiked with dog feces samples. The mean sensitivity was in between 10°62 and 10°8 FAID50 per reaction for different CPV types (table 3). However, the cycle sequencing reaction required >103 FAID50 virus to offer an unambiguous sequencing result. Since, only the pertinent SNPs useful in differentiating the types were involved in the mini- sequencing reaction, the result analysis will be simpler and rapid compared to the tedious sequence analysis to determine the types in cycle sequencing. Therefore, the mini-sequencing method can be used to rapidly type numerous CPV samples in a single tube.

Examples 7

Specificity of mini-sequencing technique in identifying CPV types

The mini-sequencing reaction specificity was assessed by performing the reaction in genomic DNA extracted from other related canine viruses. The genomic DNA of canine adenovirus 1, canine adenovirus 2, canine distemper virus, canine caronavirus and canine parainfluenza virus were subjected to mini-sequencing reaction using CPV specific primers. None of the DNA was amplified in the PCR and did not produce any signal in the capillary gel electrophoresis indicating that the assay is specific for detecting the CPV types.

Examples 8

Correlation of mini-sequencing technique with conventional cycle sequencing in typing CPV field isolates

The CPV typing using the mini-sequencing reaction was performed for 13 CPV field isolates and two vaccine strains (CPV 2) available in our repository. These isolates were typed earlier by sequencing. Of the 13 field isolates 9 belonged to CPV2a and 4 were CPV2b. The typing results obtained from mini-sequencing matched 100% with that of cycle sequencing.

SEQ ID NO: 8 shows nucleotide sequence of CPV2a (DQ182624) (576 nucleotides)

Table 1: Sequences of the primers used for PCR amplification and mini-sequencing reaction are provided. The amino acids specific for each type, the resultant SNPs and their positions are also provided. The typing primers are one base short of the respective SNP positions either in the sense strand or in the anti-sense strand as indicated.

Table 2: The typical mini-sequencing signature for each of the CPV types and FPV is provided below. The expected size of the primers was determined because the added labeled ddNTP varies the primer mobility in capillary gel considerably.


* Anti-sense primers add complementary base; position of the SNPs in the mini-sequencing was based on the size of the primer used for the particular SNP identification. Thus, the SNP arrangements in the mini-sequencing were not matching their position in the genome.

Table 3: Mean sensitivity of the mini-sequencing technique was determined using plasmid constructs containing CPV VP2 and dog feces samples spiked with known titer of virus. The assay was performed in triplicates and repeated on three different days. The mean ± SD (N=9) for each of the types is provided in the table.

I/We Claim:

1. A set of oligonucleotides for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) types in a sample, wherein the set comprises oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 13.

2. The set of oligonucleotides as claimed in claim 1, wherein the Canine Parvovirus (CPV) type is selected from a group consisting of CPV2, CPV2a, CPV2b and CPV2c.

3. A kit for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) type in a sample, wherein the kit comprises the set of oligonucleotides as claimed in claim 1, dNTPs, ddNTPs, polymerase and reaction buffer.

4. A process for detection, and differentiation of Feline Panleukopaenia Virus (FPV) and Canine Parvovirus (CPV) types in a sample, wherein the process comprises

a. providing a sample potentially containing at last one target nucleic acid sequence;

b. performing a polymerase chain reaction using a reaction mixture comprising the sample and the oligonucleotides as set forth in SEQ ID NO: 6 and SEQ ID NO: 7 to obtain an amplified product;

c. performing a mini-sequencing reaction using a mini-sequencing reaction mixture comprising the amplified product, and the oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 13 to obtain mini-sequencing product;

d. separating the mini-sequencing products by size or electrophoretic mobility; and

e. detecting the presence of the target nucleic acid sequence by distinguishing the separated mini-sequencing product wherein the target nucleic acid sequence is of FPV, CPV2, CPV2a, CPV2b or CPV2c.

5. The process as claimed in claim 4, wherein size of the amplified product of step (b) is 576 bp.