Multiplex PCR primer panel and kit for detection and genotyping of lumpy skin disease virus

Abstract

The present invention relates to a highly sensitive nested multiplex PCR-based primer panel for the detection of Lumpy Skin Disease Virus (LSDV) in a biological sample obtained from bovine species and livestock animals In particular, the present invention provides a nested multiplex detection primer panel and a kit comprising the same, for the detection of Lumpy Skin Disease Virus (LSDV) and its variants thereof. The present invention also provides a multiplex primer panel for the PCR whole genome amplification and genotyping of Lumpy Skin Disease Virus (LSDV) to obtain genomic data, which can be useful in tracking the evolution of the Lumpy Skin Disease virus and correlating with disease severity. Therefore, the present invention emphasizes its utility in veterinary diagnostics, epidemiological studies, and disease management strategies related to vaccine development for Lumpy Skin Disease Virus (LSDV) infections in livestock.

Description

[0001] MULTIPLEX PCR PRIMER PANEL AND KIT FOR DETECTION AND GENOTYPING OF LUMPY SKIN DISEASE VIRUS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to primers, primer panels and kits for detection of Lumpy Skin Disease Virus (LSDV). The present invention also relates to a whole genome amplification panel for genotyping of Lumpy Skin Disease Virus (LSDV) in a sample.

[0004] BACKGROUND AND PRIOR ART OF THE INVENTION

[0005] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the present invention, or that any publication specifically or implicitly referenced is prior art.

[0006] Bovine Lumpy Skin Disease (LSD) is caused by the Lumpy Skin Disease Virus (LSDV), a pox virus of the Capripoxvirus genus and Poxviridae family. The genus also includes the goat pox and sheep pox viruses, and together they cause a significant health impact on domestic ruminants. LSDV is an enveloped, linear double- stranded DNA virus, with -151 kbp genome containing 2.4 kbp inverted terminal repeats at the ends. The viral genome is annotated with 156 putative genes, encoding structural and non-structural proteins, which are highly conserved and antigenically similar in the Capripox virus genus.

[0007] LSD is a host-specific transboundary disease affecting cattle (Bos taunts, Bos indicus) and water buffalo (Bubalus bubalis), and is listed in the WOAH (World Organization for Animal Health) website as notifiable disease (https: / / www.woah.org / en / disease / lumpy-skin-disease / ). Although experimental infections in impala and giraffe have shown clinical symptoms, there is no evidence of natural infection in these animals. Recent reports of LSDV in Gazella bennettii in Rajasthan, India, suggest potential host range expansion, posing a significant threat to both domestic and wildlife species. Although LSD is classified as non-zoonotic, transmission to humans cannot be ruled out. LSDV was detected in samples from human subjects suspected of infection in a study by the Animal Health Research Institute (AHRI), Egypt.

[0008] LSDV transmission can occur via ticks (Rhipicephalus and Amblyomma species), biting flies (Stomoxys calcitrans), and mosquitoes (Aedes aegypti) and is facilitated by abiotic factors such as animal trade, introduction of animals sourced from outside the herd, shared feed or water sources, vertical transmission, and nursing from infected udders. LSDV can remain viable in the environment under optimal conditions, which can facilitate transmission of the virus and spread of disease. While cattle of all ages, different breeds and both sexes are susceptible to LSDV, lactating cows, young calves, and underweight cattle show higher vulnerability. Clinical symptoms of LSD, including the prominent skin nodules, are well documented and disease morbidity ranges from 3% to 85%, while mortality is reported at < 10%. LSDV was initially reported from Zambia and South Africa in the 1920s and is now known to be prevalent across Africa, the Middle East, Europe, and Asia. In India, LSDV was first reported in 2019 to affect livestock of marginal farmers in Odisha state with 7% morbidity and no mortality. However, by 2020, LSDV infections turned fatal, heavily impacting the northwest regions of Rajasthan, Gujarat, and Maharashtra states, and by 2022, ~2.5 million cases and over 100,000 cattle deaths were recorded in India. As India has the world’s largest dairy cattle population the economic impact of LSD on its dairy industry is substantial.

[0009] Traditional diagnostic methods for LSDV include serological assays, viral isolation, immunohistochemistry, and conventional PCR. Serological assays, such as ELISA and indirect fluorescent antibody tests, are useful for detecting antibodies against LSDV but cannot distinguish between active and past infections. Viral isolation is considered the gold standard for diagnosis, but it is time-consuming, labour-intensive, and requires specialised biosafety facilities. Immunohistochemistry enables visualization of viral antigens in tissue samples but is limited by the need for high-quality specimens and laboratory infrastructure. Conventional PCR and real-time PCR have improved the sensitivity and specificity of LSDV detection, but these methods may be affected by genetic variation in the viral genome, potentially leading to false negatives.

[0010] Gamil et al. (Vet World. 2019 Jul; 12(7): 1093-1100) describe a study in Egypt where detection of Lumpy Skin Disease Virus (LSDV) in cattle was done using real-time PCR and serological assays across various regions. LSDV-positive samples were detected by RT-PCR, followed by conventional PCR and then FAT. The indirect ELISA detected more antibody-positive samples than the IF AT from cattle serum samples.

[0011] Amin et al. (Vet World. 2021 Aug; 14) on naturally occurring lumpy skin disease virus (LSDV) in cattle employed virological, molecular, and immunohistopathological assays for diagnosis. The virus was isolated into the chorioallantoic membrane (CAM) of embryonated chicken eggs ECEs, with 58 of 73 samples showing pock lesions on the CAM. Twenty-two skin specimens were used for molecular, histopathological, and IHC diagnosis. Conventional PCR was used to detect LSDV DNA in all samples, with IHC confirming the antigen in epidermal and hair follicle cells. The paper suggests that while viral isolation is the gold standard, it's time-consuming. IHC is crucial for visual antigen detection, especially in labs lacking advanced biosafety facilities.

[0012] Alexander Sprygin et. al (BMC Research Notes volume 16, 2023, Article number: 247) describes the development and implementation of a real-time PCR assay specifically designed to detect LSDV. The assay aims to improve diagnostic capabilities by offering high sensitivity and specificity compared to conventional methods. Key aspects of the research include the optimization of PCR primers for LSDV detection and the validation of the assay using clinical samples. The application of this real-time PCR assay is highlighted in various settings, emphasizing its utility in veterinary diagnostics, epidemiological studies, and disease management strategies related to LSDV infections in livestock.

[0013] Yadav et. al (BMC Genomics, 2024, 25: 196) studied biological specimens, including saliva swabs, blood samples, and skin lesion scabs, collected from infected cattle from different regions of India. For the detection of LSDV, conventional PCR was performed to get a PCR amplicon of 472 bp in size. Recent studies have revealed substantial genetic diversity among LSDV isolates, particularly in India, where novel mutations and genotypes have emerged. This genetic variation poses challenges for accurate detection, surveillance, and vaccine efficacy assessment. Furthermore, the rapid spread of LSDV and its impact on the dairy industry underscore the need for robust, sensitive, and scalable molecular diagnostic tools that can be deployed in field settings for large-scale surveillance and genotyping.

[0014] Current approaches often rely on single-target PCR assays or serological tests, which may not provide comprehensive information about viral diversity or detect low-level infections in asymptomatic animals. There is a clear need for improved molecular methods that enable multiplexed detection of LSDV, resilience to genetic variation, and the ability to perform wholegenome sequencing for genotyping and epidemiological studies.

[0015] There is need to develop methods that can help address this requirement, and may also support early diagnosis, improve monitoring of viral evolution, and strengthen disease surveillance strategies for LSDV in India.

[0016] OBJECTIVES OF THE INVENTION

[0017] Accordingly, the main objective of the present invention is to develop methods to detect Lumpy Skin Disease Virus (LSDV) and methods to amplify the whole genome of LSDV from biological samples, followed by genome sequencing.

[0018] Another objective of the present invention is to provide a feasible, robust and easy-to-implement method to enable routine surveillance of the virus.

[0019] Another objective of the present invention is to provide a feasible, robust and easy-to-implement method for the genome sequencing of LSDV.

[0020] Another objective of the present invention is to provide a feasible, robust and easy-to-implement method for non-invasive sample collection, storage, and a rapid diagnostic technique for the detection of Lumpy Skin Disease Virus (LSDV) in cattle using a highly sensitive multiplex nested PCR technique.

[0021] Yet another objective of the present invention is to provide a method that can more effectively detect and genotype Lumpy Skin Disease (LSDV) in symptomatic or asymptomatic bovine samples.

[0022] Still another objective of the present invention is to provide a primer panel comprising a set of unique primers with an optimized method for performing nested multiplex PCR to detect LSDV (namely LSDV_NMDPP) in the symptomatic or asymptomatic bovine samples.

[0023] Yet another objective of the present invention is to provide a rapid, sensitive, and improved nested multiplex polymerase chain reaction (PCR) employing the said primer panel for detecting LSDV Lumpy Skin Disease Virus and its variants in symptomatic or asymptomatic bovine samples.

[0024] Still another objective of the present invention is to provide a kit for detecting LSDV, Lumpy Skin Disease Virus, in symptomatic or asymptomatic bovine samples.

[0025] Yet another objective of the present invention is to provide a primer panel (LSDV_WGSPP_3.5) for multiplexed PCR amplification and sequencing of the LSDV Lumpy Skin Disease Virus genome for identifying viral variants in biological samples for disease surveillance studies. SUMMARY OF THE INVENTION

[0026] Accordingly, the present invention provides a primer panel for genome amplification of Lumpy Skin Disease Virus (LSDV). The said primer panel can have pools of primers, each pool comprising

[0027] (i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30,

[0028] 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81,

[0029] 82, 85, 86, 89, 90, 93, 94 or variants thereof ; and

[0030] (ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32,

[0031] 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83,

[0032] 84, 87, 88, 91, 92, 95, 96 or variants thereof.

[0033] The present invention relates to a composition for genome amplification of Lumpy Skin Disease Virus (LSDV). The said composition can comprise the primer panel of the present invention. It can optionally comprise a master mix containing dNTPs, buffer, and a DNA polymerase.

[0034] The present invention relates to a primer set for the detection of Lumpy Skin Disease Virus (LSDV).

[0035] The said primer set can comprise

[0036] (i) a set of outer primers having SEQ ID NOs: 97-102 or variants thereof; and

[0037] (ii) a set of inner primers having SEQ ID NOs: 103-108 or variants thereof.

[0038] The present invention relates to a method for the detection of Lumpy Skin Disease Virus (LSDV) in a sample by nested multiplex PCR. The method comprising the steps of contacting denatured target DNA in the sample with a primer set comprising outer primers of SEQ ID NO: 97-102 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to produce a first PCR product and contacting said first PCR product with a primer set comprising inner primers of SEQ ID NOs: 103-108 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to produce the amplified copies of nucleic acids are obtained.

[0039] The method involves primary and secondary PCR cycles, the primary PCR cycle comprising: initial denaturation at 70-98 °C for 3-7 minutes, followed by one or more cycles of 70-98 °C denaturation for 20-40 seconds; annealing at 40-70 °C for 20-40 seconds; extension at 65-80 °C for 1-3 minutes, followed by final extension at 65-80 °C for 2-5 minutes and the secondary PCR cycle comprising: initial denaturation at 70-98°C for 3-7 minutes, followed by one or more cycles of 70-98°C, preferably at 94°C denaturation for 20-40 seconds; annealing at 40- 70°C, preferably at 55°C for 20-40 seconds; extension at 65-80 °C for 1-2 minutes, followed by final extension with at 65-80 °C, preferably at 72 °C for 1-3 minutes.

[0040] The present invention relates to a method for the whole genome sequencing of the Lumpy Skin Disease Virus (LSDV) by multiplex polymerase chain reaction (PCR), wherein the method comprises mixing half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94 or variants thereof , a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product, mixing the other half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83, 84, 87, 88, 91, 92, 95, 96 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product, processing the PCR products thus obtained for library preparation for sequencing.

[0041] The multiplex PCR method comprises: denaturation at 90-99 °C for 20-40 seconds, followed by one or more cycles of 90-99 °C denaturation for 10-40 seconds, preferably, at 98 °C; annealing and extension at 40-70 °C, preferably 65 °C.

[0042] The present invention relates to amplicons in each of the PCR products that can have a size of about 3.5 kb, with overlapping regions of about 0.5 kb. The sample can be a bovine sample.

[0043] The present invention relates to a kit for the detection of Lumpy Skin Disease Virus (LSDV). The kit can comprise,

[0044] (i) a set of outer primers having SEQ ID NOs: 97-102 or variants thereof;

[0045] (ii) a set of inner primers having SEQ ID NOs: 103-108 or variants thereof;

[0046] The present invention provides a kit for the amplification of Lumpy Skin Disease Virus (LSDV) genome. The kit can comprise

[0047] (i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30,

[0048] 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81,

[0049] 82, 85, 86, 89, 90, 93, 94 or variants thereof; and

[0050] (ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32,

[0051] 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83,

[0052] 84, 87, 88, 91, 92, 95, 96 or variants thereof;

[0053] The kit can further comprise,

[0054] (i) deoxynucleotide triphosphates (dNTPs),

[0055] (ii) a buffer,

[0056] (iii) DNA polymerase,

[0057] (iv) optionally, a positive control containing synthetic DNA segments of amplification targets,

[0058] (v) optionally, a negative control containing a reaction mixture without any nucleic acid,

[0059] (vi) nuclease-free water.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1: shows the genomic coordinates for the target region of the three nested PCRs.

[0062] Figure 2: shows the gel image indicating the presence of -500 bp fragment obtained from the diagnostic multiplexed nested PCR reaction.

[0063] Figure 3: represents the PCR assays showing the analytical sensitivity of the multiplexed nested PCR.

[0064] Figure 4: represents the primer panel (LSDV_WGSPP_3.5 primer set) showing pool A and pool B overlap PCR amplicons across LSDV genome.

[0065] Figure 5: shows the PCR products obtained using the primer panel (LSDV_WGSPP_3.5 primer set) used for the amplification of the entire LSDV genome as -3.5 kb fragments. Figure 6: shows read Depth plots for nanopore sequence data of LSDV genomic fragments obtained by multiplexed amplification: The LSDV genome was amplified by multiplexed PCR using two different primer panels, LSDV_WGSPP_3.5 (A & C) and LSDV_WGSPP_7.5 (B). Sample PP488409 was evaluated, using two different Taq DNA polymerases, Quantabio replica hifi tough mix (A & B) or Takara primestar GXL master mix (C), for PCR amplification. The resolution of the plot is at the nucleotide level, and red marks indicate the positions at which the read depth was <20X. The quality of the nanopore sequence data obtained with the LSDV_WGSPP_3.5 primer panel multiplexed PCR using Quantabio replica hifi tough mix was suitable for variant calling.

[0066] Figure 7: Phylogenomic relationship of LSDV field strains with previously reported isolates. Maximum likelihood phylogeny was performed for the LSDV genomes sequenced in this study (41 field samples) and 133 other global LSDV genomes. The resulting tree file was used to render an unrooted tree structure. The sequences reported from India are grouped in clade 1.2.1.3. The geographic region from which the viruses were reported is shown. The clade branches are colour- coded, and the major clades are labelled as previously reported. Clade 1.2.2 includes NC_003027.1 and clade 1.1 contains the African Neethling vaccine strains.

[0067] DETAILED DESCRIPTION OF THE INVENTION:

[0068] The tables, figures and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

[0069] As used herein, a sample refers to any substance containing or presumed to contain nucleic acid and includes a sample isolated from cattle.

[0070] As used herein, the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” refer to primers, oligomer fragments to be detected, and oligomer controls.

[0071] As used herein, the term “Primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method.

[0072] As used herein, the term “amplification” refers to methods to increase the number of copies of a nucleic acid molecule. The resulting amplification products are typically called “amplicons.” Amplification of a nucleic acid molecule (such as a DNA or RNA molecule) refers to the use of a technique that increases the number of copies of a nucleic acid molecule (including fragments). In some examples, an amplicon is a nucleic acid from a cell, or acellular system, such as mRNA or DNA that has been amplified.

[0073] As used herein, the term “nucleoside triphosphate” is used herein to refer to nucleosides present in either DNA or RNA and thus includes nucleosides which incorporate adenine, cytosine, guanine, thymine and uracil as bases, the sugar moiety being deoxyribose or ribose. Embodiments of the present disclosure relate to a nested multiplex PCR-based detection method for disease surveillance. In particular, the present disclosure provides a rapid and sensitive nested multiplex primer panel (NMPP) to detect Lumpy Skin Disease Virus (LSDV) from the given sample, and a kit comprising the same. The present invention also provides a primer panel for multiplexed PCR amplification (WGSPP) of the whole genome of the LSD virus for genotyping and identifying viral variants.

[0074] The present invention can provide compositions, methods, and kits for the rapid, sensitive, and robust detection and genotyping of Lumpy Skin Disease Virus (LSDV) in bovine samples using nested multiplex PCR and whole genome sequencing approaches. The invention addresses the urgent need for improved molecular tools for LSDV surveillance, especially in regions where the disease is endemic and genetic variation among viral strains is high.

[0075] The present invention can provide a nested multiplex detection primer panel (NMDPP) comprising a set of unique primers (SEQ ID NOs: 97-108) for the sensitive detection of LSDV in symptomatic or asymptomatic bovine samples. The NMDPP can target three distinct regions (Figure 1) of the LSDV genome, ensuring robust detection even in samples with low viral load or degraded DNA. The invention also provides a kit comprising the NMDPP, master mix containing deoxynucleotide triphosphates (dNTPs), buffer, DNA polymerase, positive and negative controls, and nuclease-free water for in vitro detection of LSDV by nested multiplex PCR.

[0076] The invention can further provide optimized protocols for non-invasive sample collection (e.g., skin swabs, nasal swabs), DNA extraction, PCR setup, and sequencing library preparation. The methods are compatible with portable thermocyclers and field-deployable equipment, making them suitable for large-scale surveillance and rapid diagnostics in resource-limited settings.

[0077] The compositions and kits of the invention can be validated for high sensitivity, may be capable of detecting about >30 copies of LSDV DNA, and can be resilient to genetic variation due to multiplexed targeting of conserved regions. The bioinformatics pipeline for sequence data analysis may include base calling, demultiplexing, quality filtering, mapping to the reference genome, primer trimming, variant calling, consensus sequence generation, depth masking, variant annotation, sequence alignment, and phylogenetic tree construction.

[0078] The present invention can provide an end-to-end solution for the detection and genotyping of LSDV in bovine samples, enabling rapid, sensitive, and cost-effective surveillance and disease management. The invention may be advantageous for field deployment, large-scale epidemiological studies, and monitoring of vaccine efficacy in regions affected by LSDV.

[0079] In an embodiment, the present invention provides a primer set for the detection of LSDV (Table 1). The primer set can comprise:

[0080] • Outer primers: SEQ ID NOs: 97-102 (or variants thereof)

[0081] • Inner primers: SEQ ID NOs: 103-108 (or variants thereof)

[0082] For illustration purpose, a nested multiplex PCR may be performed using the outer primers for the first round of amplification, followed by the inner primers for the second round, increasing sensitivity and specificity for LSDV detection.

[0083] In an embodiment, the present invention provides a method for detecting LSDV in a sample by nested multiplex PCR. The method can comprise the step of contacting denatured target DNA with the outer primers and PCR reagents to produce a first PCR product. The first PCR product can be used as a template for a second PCR with the inner primers and PCR reagents to obtain amplified nucleic acids.

[0084] For example, a nasal swab from a cow can be processed for DNA extraction. The nested multiplex PCR can be performed as described, and the presence of a -500 bp band on agarose gel may confirm LSDV infection (Figure 2).

[0085] Field samples from infected cattle may be processed using this method, and the resulting detection data may be used to track outbreaks and surveillance studies.

[0086] The method may be performed using a bovine sample, such as a skin swab, nasal swab, blood, or lesion scab.

[0087] In another embodiment of the present invention, the biological sample used for the detection may be obtained from domestic cattle (e.g. Bos taunts, Bos indicus) or water buffalo (e.g. Bubalus bubalis).

[0088] In yet another embodiment of the present invention, the biological sample may be a bovine sample, such as a skin swab, nasal swab, blood, or lesion scab etc. The sample may be clinical sample directly from cattle i.e. skin swab, nasal swab, milk, blood or lesion swab etc., or environmental samples involving drinking water, waste water, soil or feed etc., from cattle farms.

[0089] In an embodiment of the present invention, the extension is performed by polymerase chain reaction (PCR). The extension or thermocycling parameters can also be varied to take advantage of the differential thermal stability between the forward primer and reverse primer. For example, following the denaturation step in thermocycling, an intermediate temperature may be introduced, which is permissible for enriching primer binding but not for amplification primer binding; the temperature is then increased to the extension temperature (for example, 72° C), whereby permitting extension of the matched enriching primer and melting away of unextended enriching primer. The cycles of an intermediate temperature and extension temperature can be repeated as many times as desirable to allow the matched primer to extend on as many target templates as possible. The temperature can then be reduced to permit amplification primer annealing and extension.

[0090] Yet another embodiment of the present invention provides a method comprising a primary PCR cycle, wherein the outer and inner primers are added in their respective tubes, followed by NMPCR reaction consisting of denaturation, annealing, and extension / elongation amplification reactions, followed by a secondary PCR cycle comprising steps similar to the primary PCR cycle.

[0091] Primary PCR Cycle:

[0092] (i) The initial denaturation are effected at 70-98 °C for 3-7 minutes, followed by 20 cycles of 70-98 °C denaturation for 20-40 seconds. Preferably, the temperature may be 94 °C.

[0093] (ii) the annealing is effected at 40-70 °C for 20-40 seconds. Preferably, the temperature may be 50 °C.

[0094] (iii)the extension is effected at 65-80 °C for 1-3 minutes, followed by final extension with at 65- 80 °C for 2-5 minutes. Preferably, the temperature may be 72 °C.

[0095] Secondary PCR Cycle: (i) The initial denaturation and denaturation are effected at 70-98 °C for 3-7 minutes, followed by 25 cycles of 70-98 °C denaturation for 20-40 seconds. Preferably, the temperature may be 94 °C.

[0096] (ii) the annealing is effected at 40-70 °C for 20-40 seconds. Preferably, the temperature may be 55 °C.

[0097] (iii)the extension is effected at 65-80 °C for 1-2 minutes, followed by final extension with at 65- 80 °C for 1-3 minutes. Preferably, the temperature may be 72 °C.

[0098] In an embodiment of the present invention, the amplification reaction is selected from any of the polymerase chain reactions (PCRs) known in the prior art.

[0099] Still another embodiment of the present invention, wherein our Nested Multiplex Detection PCR (NMDPP) panel could detect about 0.1 fg of viral DNA corresponding to containing about 30 or more copies of LSDV consistently producing a bright amplification band on gel (e.g. Figure 3) in a multiplexed PCR reaction, hence confirming its high analytical sensitivity.

[0100] Yet another embodiment of the present invention, wherein the nucleotide sequence of the NMPCR- amplified sequence can be determined using anyone or combination of the variety of methods for determining the nucleotide sequence of DNA. Preferably, the method may be Oxford Nanopore Technology (ONT).

[0101] In an embodiment of the present invention, the NMDPP targets different regions of the LSD viral genome for the diagnosis of positive samples, even with degraded DNA. This method is sensitive enough to detect the virus with as few as 30 copies in the sample.

[0102] In another embodiment, the detection of the target virus is possible even when the sample contains a partially degraded viral genome, as three different locations are targeted for detection, and the method is more resilient to genetic variations. Furthermore, because of multiple target regions, detection is possible even if a mutation occurs in one of the primer binding sites.

[0103] In an embodiment of the present invention, the NMDPP designed for swift and reliable virus detection, may prove valuable in disease surveillance, especially during seasonal outbreaks, and monitoring vaccine efficacy at a nominal cost. Moreover, it can serve as a source for the detection of the virus in asymptomatic and early-stage disease animals, which is very much needed for disease control and stopping the spread of the virus.

[0104] In an embodiment of the present invention, the NMDPP has been developed for bovine sample diagnostics of Lumpy Skin Disease Virus (LSDV), referenced from NCBI (NC_003027.1 Lumpy Skin Disease virus NL2490), wherein said method uses three distinct nested PCR amplicons, strategically located at the 5’ end, in the middle, and towards the 3’ end of the viral genome (Figure 1) to ensure PCR amplification even if the viral genome copies in the sample were not intact or degraded or mutated and to maximise the specificity of the test.

[0105] In an embodiment, the present invention provides a primer panel for the genome amplification of Lumpy Skin Disease Virus (LSDV). The panel may comprise the following pools:

[0106] • Pool A: Comprises primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 93, 94 (or variants thereof). • Pool B: Comprises primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83, 84, 87, 88, 91, 92, 95, 96 (or variants thereof).

[0107] The present invention provides a primer panel (LSDV_WGSPP_3.5) comprising 48 overlapping primer pairs (SEQ ID NOs: 1-96) for multiplexed PCR amplification of the entire LSDV genome, which may be used in the whole genome sequencing of LSDV. For illustration purpose, the panel may be divided into Pool A and Pool B as mentioned above, each used in a separate multiplex PCR reaction to amplify ~3.5 kb fragments with 0.5kb overlaps covering 99.8% of the LSDV genome (Figure 4). The PCR products may be pooled and sequenced using high-throughput platforms such as Oxford Nanopore Technology (ONT) or any other technology known to a person skilled in the art, enabling rapid, high-coverage amplification and sequencing. This facilitates surveillance of viral variants, monitoring of vaccine efficacy, and epidemiological studies.

[0108] In the present invention, a sample containing LSDV DNA may be split into two aliquots. Each aliquot may be amplified using either Pool A or Pool B primers in a multiplex PCR reaction, generating overlapping amplicons that cover nearly the entire viral genome.

[0109] The present invention may provide a composition for genome amplification of LSDV. The composition may comprise the primer panel of the present invention. The composition may further comprise a master mix containing dNTPs, buffer, and a DNA polymerase.

[0110] The composition may be used to amplify LSDV DNA from a bovine sample (eg. skin swab or nasal swab). The resulting PCR products can be suitable for downstream sequencing or genotyping.

[0111] In a preferred embodiment, the LSDV_WGSPP_3.5 primer set of the present invention may amplify shorter 3.5 kb genomic amplicons with uniform efficiency, covering 99.8% of the LSDV genome.

[0112] In an embodiment, the primer panel of the present invention may comprise 48 overlapping amplicons of ~3.5 kb size covering the entire genome.

[0113] In an embodiment, the present invention can provide a method for whole genome sequencing of LSDV by multiplex PCR. The method can comprise dividing the sample into two halves, each mixed with either Pool A or Pool B primers and PCR reagents. The DNA can be amplified in two separate reactions. The PCR products can be pooled and processed for library preparation and sequencing. The sequence may be performed in such a manner that each PCR product amplicon can be approximately 3.5 kb in size, with overlapping regions of about 0.5 kb. This design may ensure complete genome coverage and may facilitate accurate assembly of the viral genome from sequencing data. Field samples from infected cattle may be processed using this method, and the resulting sequencing data may be used to identify viral variants and track outbreaks.

[0114] The method may be performed using a bovine sample, such as a skin swab, nasal swab, blood, or lesion scab etc. The sample may be clinical sample directly from cattle i.e. skin swab, nasal swab, milk, blood or lesion swab etc., or environmental samples involving drinking water, waste water, soil or feed etc, from cattle farms.

[0115] In an embodiment of the present invention, the LSDV_WGSPP_3.5 has been developed for bovine sample genotyping of Lumpy Skin Disease Virus (LSDV), wherein said method uses 48 distinct PCR amplicons, strategically separated as pool A and pool B primer sets containing 48 primers each (Figure 4) to ensure PCR amplification of the LSDV whole genome from cattle samples.

[0116] In an embodiment of the present invention, the PCR is the two-step reaction with annealing and extension at the same temperature and same step, with the initial denaturation during the PCR is effected at 94-98 °C for 20-30 seconds, followed by 35 cycles of 70-95 °C denaturation for 10- 20seconds and 60-68 °C annealing and extension for 4-6 minutes. Preferably, the temperature is 98 °C for denaturation and 65 °C for annealing and extension, respectively.

[0117] In some embodiments, the mutations are selected from but limited to non-synonymous mutations, synonymous mutations, insertions or deletions (indels) distributed in coding and / or non-coding regions. The bioinformatics pipeline for sequence data analysis may include base calling, demultiplexing, quality filtering, mapping to the reference genome, primer trimming, variant calling, consensus sequence generation, depth masking, variant annotation, sequence alignment, and phylogenetic tree construction.

[0118] In an embodiment of the present invention, the mutations may be found in genes encoding viral virulence factors, including but not limited to kelch-like protein (LSDV019), putative host range protein (LSDV067) and gene with unknown function (LSDV026), wherein the mutations are multiple mutations.

[0119] In an embodiment of the present invention, the mutations are indels found in the coding region of three genes (position 13,103 in LSDV019, position 18,174 in LSDV026 and position 80,822 in LSDV087) that result in truncation of the coding sequence and likely result in disruption of gene functions.

[0120] In an embodiment of the present invention, the mutations may be commonly occurring mutations enriched in the genes with viral virulence and host range function selected from but not limited to LSDV005, LSDV019, LSDV067, LSDV128, LSDV144, LSDV150, and LSDV151.

[0121] The PCR conditions have been extensively optimised (including adjustment of primer concentrations) and may provide the best results with good whole-genome coverage for in-depth analysis.

[0122] Yet another embodiment of the present invention provides a method comprising a PCR cycle, wherein the Pool A and Pool B are added in their respective tubes, followed by a whole genome amplification PCR reaction consisting of initial denaturation, denaturation, annealing and extension / elongation as a single step amplification reactions,

[0123] Whole genome amplification PCR cycle:

[0124] (i) The initial denaturation are effected at 90-99 °C for 20-40seconds, followed by 35 cycles of 90-99 °C denaturation for 10-40 seconds. Preferably, the temperature may be 98 °C.

[0125] (ii) the single-step annealing and extension is effected at 40-70 °C for 6 minutes. Preferably, the temperature may be 65 °C.

[0126] In an embodiment of the present invention, the amplification reaction is selected from any of the polymerase chain reactions (PCRs) known in the prior art. In an embodiment the present invention can provide a kit for the detection of LSDV. The kit can comprise

[0127] • Outer primers: SEQ ID NOs: 97-102 (or variants thereof)

[0128] • Inner primers: SEQ ID NOs: 103-108 (or variants thereof)

[0129] The kit may be supplied with instructions and reagents for nested multiplex PCR, enabling rapid field or laboratory detection of LSDV.

[0130] In an embodiment of the present invention, the DNA polymerase may be selected from, but not limited to, EmeraldAmp GT PCR Master Mix, Takara or GoTaqGreen Master Mix, Promega. This corresponds to the detection of 30 copies or more of the viral DNA molecule and demonstrates the high sensitivity of the nested multiplexed PCR assay developed in this study for LSDV detection from field samples.

[0131] In an embodiment, the present invention can provide a kit provided for whole genome sequencing of LSDV. The kit can comprise

[0132] . Primers selected from SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 93, and 94

[0133] . Primers selected from SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83, 84, 87, 88, 91, 92, 95, and 96

[0134] The kit may be used to perform multiplex PCR for whole genome amplification of LSDV from field samples, enabling downstream sequencing and genotyping. The multiplex whole genome amplification PCR for LSDV can be performed as described, and the presence of a -3500 bp band on agarose gel may confirm LSDV genome amplification (Figure 5).

[0135] The kit can further comprise of deoxynucleotide triphosphates (dNTPs), buffer, DNA polymerase, optionally, a positive control containing synthetic DNA segments of amplification targets, and optionally, a negative control containing a reaction mixture without any nucleic acid or nuclease- free water.

[0136] The kits of the present invention may be provided as a ready-to-use package for veterinary diagnostic labs, including all necessary reagents and controls for reliable LSDV detection and genome amplification.

[0137] In another invention, the LSDV_WGSPP_3.5 primer panel (Table 6) is used to amplify the whole genome of the virus and identify the LSDV variants that comprise one or more mutations in the LSDV genome. Another embodiment ensures vaccine preparedness for changing variants and mitigating the risk of zoonotic potential of the disease.

[0138] In an embodiment of the present invention, the DNA polymerase is selected from but not limited to Quantabio repliQa HiFiToughMix, Takara PrimeSTAR GXL, NEB Q5 Hot Start High-Fidelity 2X Master Mix, and NEB LongAmp Hot Start Taq DNA Polymerase (Figure 6). This corresponds to the genotyping of the LSDV template DNA in the sample.

[0139] In some embodiments of the present invention, the methods and compositions described herein may be used for detecting LSDV and other capripoxviruses, such as sheep pox virus and goat pox virus. In some embodiments of the present invention, the methods and compositions described herein may be used for detecting LSD virus target molecules including, but not limited to, mRNA, rRNA, tRNA, RNA, DNA, or combinations thereof, wherein the nucleic acid comprises a target nucleotide sequence that may be used to distinguish among pathogenic species / strains from one another.

[0140] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

[0141] EXAMPLES

[0142] The invention is further illustrated by the following examples, which should not be construed to limit the scope of the invention in any way.

[0143] Materials:

[0144] Chemicals:

[0145] 1. Pooled primer sets for both LSDV_NMDPP and LSDV_WGSPP_3.5 panels (options recommended are from Integrated DNA Technologies (IDT))

[0146] 2. Taq DNA Polymerase (options recommended are repliQa HiFiToughMix supplied by Quantabio and EmeraldAmp GT PCR Master Mix supplied by Takara)

[0147] 3. Positive control template

[0148] 4. Sample DNA template

[0149] 5. Nuclease-free water

[0150] 6. Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) OR Rapid Barcoding Kit 96 V14 (SQK- RBK114.96) for sequencing.

[0151] In an embodiment of the present invention, the primer sets are selected from but not limited to Integrated DNA Technologies (IDT), Barcode Biosciences (BBS), etc. This corresponds to the starting material for the amplification of the target required for the detection and genotyping of the LSDV template DNA in the sample.

[0152] The biological materials used in the present invention, such as cattle nasal swabs and cattle skin scab swabs, were procured from Indian farm animals. Collection of all field samples was conducted with the explicit consent of cattle owners in compliance with guidelines set by regulatory bodies.

[0153] Example 1

[0154] Sample collection and DNA extraction:

[0155] Samples were collected from dairy cows from Pune and Ahmednagar different districts of Maharashtra state, and Mayurbhanj and Angul districts of Odisha state. Sample collection was authorized by the animal ethics and biosafety committees of BAIF Development Research Foundation, Pune. Samples were acquired from animals in 2022 and 2023 when LSDV outbreaks and transmission was occurring in the sample collection sites. Diseased animals were selected for sample collection based on the presence of characteristic skin nodules, which had turned into skin lesions. In addition, samples were collected from apparently normal animals to assess the feasibility of early detection or asymptomatic presence of the virus. A total of 123 samples were collected non- invasively, out of which 75 were skin scab samples (from symptomatic animals) and 48 were nasal samples (from symptomatic and asymptomatic animals). DNA extraction was carried out using MagNA Pure 96 Automation System (Roche diagnostics) by following the manufacturer's guidelines.

[0156] Example 2

[0157] Nested PCR using Multiplex Detection Primer Panel (LSDV_NMDPP) A nested multiplex PCR method was developed for the detection of Lumpy Skin Disease Virus (LSDV). The design of PCR primers is based on the reference genome of LSDV available in NCBI database (NC_003027.1 Lumpy Skin Disease virus NL2490). Three distinct genomic locations were chosen for nested PCR amplification, strategically located towards the 5’ end, in the middle, and towards the 3’ end of the viral genome (Figure 1). Because this method included three nested PCRs multiplexed together, it can be used for detection of even partially degraded LSDV DNA present in the sample. Moreover, it overcomes detection failure due to primer drop-out (which can happen if primer binding region is mutated or deleted), which is expected in case of detection based on single PCR.

[0158] Details of nested PCR primer sequences are mentioned below in Table 01 below: Table 01

[0159] For individual primers, lOOpmolar stocks were prepared in Nuclease Free Water and stored in -20 °C. For multiplexed nested PCR reaction, the outer and inner primer pools were prepared by separately mixing each of the outer primers in one pool and each of the inner primers in the other pool. The primer pools can be stored in -20 °C. At the time of setting up the PCR reactions, the outer and inner primer pools were diluted 10 times (1 Opmolar primer mix) with Nuclease Free Water and added to the reactions as given below.

[0160] For nested PCR, the first PCR reaction was carried out using outer primer pool and the second PCR reaction was carried out using inner primer pool. Nested PCR setup using LSDV_NMDPP outer primer mix

[0161] Table 02 - PCR reaction mix composition using LSDV_NMDPP outer primers

[0162] Table 03 - PCR conditions for reaction using LSDV_NMDPP outer primers

[0163] The product of the first PCR using outer primer pool will amplify ~lkb region from LSDV genome.

[0164] Nested PCR setup using LSDV_NMDPP inner primers The composition of the PCR reaction mix using inner primer pool is given in Table 04

[0165] Table 04 - PCR reaction mix composition using LSDV_NMDPP inner primers

[0166] PCR amplification conditions for inner primer pool reaction is given in Table 05

[0167] Table 05 - PCR conditions for reaction using LSDV_NMDPP inner primers

[0168] The product of the second PCR using inner primer pool will amplify ~0.5kb region from the target region of LSDV genome.

[0169] Analysis of PCR products by agarose gel electrophoresis

[0170] The second PCR product was analyzed using 1% agarose gel. 5 pL of PCR product is loaded on the agarose gel and electrophoresis is carried out at 120mV for 30-40 minutes. Samples were considered positive for LSDV presence if 0.5kb band is detected. Positive samples were processed for whole genome amplification of LSDV using LSDV_WGSPP_3.5 primer panel.

[0171] Figure 2 represents the gel image showing the presence of a -500 bp fragment obtained from the diagnostic multiplexed nested PCR reaction, where M represents 100 bp plus Generuler Ladder, 1 and 2 represent the respective positive samples.

[0172] Example 3

[0173] LSDV_NMDPP sensitivity test:

[0174] To evaluate the analytical sensitivity of the nested multiplex PCR assay, the purified 1 kb fragments obtained from the outer PCRs were quantified and used for spiking 50 ng of cattle blood DNA samples at 100 fg, 10 fg, 1 fg and 0.1 fg template amounts. DNA without template spiking was used as a negative control. Template DNA copy number estimations in respective dilutions were performed using the NEBiocalculator online tool.

[0175] Figure 3 represents the image of PCR assays showing the analytical sensitivity of the multiplexed nested PCR. Lanes 1 - 3, three different negative field samples (a, b & c); Lanes 4 - 6, samples a, b & c spiked with 100 fg of 1 kb template; Lanes 7 - 9, samples a, b & c spiked with 10 fg of 1 kb template; Lanes 10 - 12, samples a, b & c spiked with 1 fg of 1 kb template; Lanes 13 - 15, samples a, b & c spiked with 0.1 fg of 1 kb template; Lanes 16 - 19, four field samples which are positive for LSDV showing the expected 500 bp multiplexed nested PCR product. Lane M - 100 bp DNA size ladder.

[0176] Example 4

[0177] Multiplexed genome amplification:

[0178] Multiplex PCR amplification of genomic segments of LSDV was developed employing a previously reported methodology by Joshua et al., for Zika and SARS-CoV-2 genome amplification and sequencing. Initially, the primer pairs reported for LSDV genome amplification by Mathijs et al., (denoted herein as LSDV_WGSPP_7.5 primer panel) were used to test their performance in the context of multiplexed PCR to give 7.5 kb amplicons covering the entire LSDV genome. The primers were mixed into two pools - pool A and pool B - to avoid amplification of shorter overlapping regions between adjacent amplicons. However, it was apparent from sequencing the multiplexed PCR product that many of the primer pairs were not working well, resulting in either total absence or a reduced number of sequence reads for the corresponding PCR fragments. Therefore, a new set of primers (denoted as LSDV_WGSPP_3.5 primer panel) was designed to amplify shorter 3.5 kb genomic amplicons with uniform efficiency, covering 99.8% of the LSDV genome. The PCR primers for the whole genome amplification of LSDV were designed using the reference genome NC_003027.1 for Lumpy skin disease virus NI-2490 available in NCBI and from preliminary sequence data generated from field samples collected in 2022 using PrimalScheme and Primer3 tools. A total of 48 primer pairs were designed to cover the entire LSDV genome and each PCR product is ~3.5kb in size with ~0.5kb overlap between adjacent PCR fragments. The LSDV_WGSPP_3.5 primer set was divided into pool A and pool B such that adjacent amplicons are in different pools. The multiplexed PCR was carried out as pool A and pool B reactions to avoid amplification of overlapping regions.

[0179] LSDV_WGSPP_7.5 primer panel from Mathijs el al. claims that it can amplify any capripoxvirus as individual PCRs covering 95% of the genome, while panel LSDV_WGSPP_3.5 of the present invention can amplify LSDV specifically in a multiplexed way with 99.8% coverage. The depth of coverage obtained from the 7.5kb panel in a multiplexed way was highly variable, and it might have over- sequenced some regions, while, the multiplexed whole genome amplification using LSDV_WGSPP_3.5 panel shows better coverage and depth when compared with the published primer panel of 7.5kb amplicons.

[0180] The detail of the entire primer set is given in Table 06.

[0181] Table 06

[0182] For individual primers, lOOpmolar stocks were prepared in Nuclease Free Water and stored in -20 °C. For the multiplexed genomic PCR reaction, the pool A and pool B were prepared by separately mixing 10 pL of each of the pool A primers in one pool and 10 pL of each of the pool B primers in the other pool. The primer pools can be stored in -20 °C. At the time of setting up the PCR reactions, the pool A and pool B primer pools were diluted 10 times (lOpmolar primer mix) with Nuclease Free Water and added to the reactions as given in Table 07 below.

[0183] Pool A and Pool B PCR reactions were set up separately using RepliQa HiFi ToughMix® Quantabio Catalogue #95200-500 master mix.

[0184] Table 07 - PCR reaction mix composition using LSDV_WGSPP_3.5

[0185] Table 08 - PCR conditions for multiplex whole genome amplification using LSDV_WGSPP_3.5 panel

[0186] Figure 5 represent PCR results obtained using LSDV_WGSPP_3.5 primer panel achieving amplification of the entire LSDV genome as ~3.5 kb fragments and ~0.5kb overlaps. Lane M - 100 bp plus DNA size ladder; lane 1 and lane 3 - Pool A PCR product of two different samples; lane 2 and lane 4 - Pool B PCR products of two different samples.

[0187] For all samples from which LSDV was detected by nested PCR, multiplexed whole genome amplification was carried out followed by Nanopore sequencing. Initially, the genome amplification was carried out using the LSDV_WGSPP7.5kb primer panel. While the production of 7.5 kb PCR fragments using this primer panel showed bands on the gel, a significant number of target regions were sub-optimally amplified in the multiplexed PCR, resulting in low coverage and depth after sequencing. To make the multiplexed genome amplification step more robust and efficient, LSDV_WGSPP3.5kb primer panel was designed and demonstrated to work efficiently in multiplexed PCR (gel image in Figure 5).

[0188] The optimal performance of the multiplexed genomic amplification was also influenced by the type of Taq DNA polymerase used. In this study, different Taq DNA polymerases reagents, such as Quantabio repliQa HiFi ToughMix polymerase, Takara Bio PrimeSTAR GXL Premix, NEB Q5 Hot Start High-Fidelity 2X Master Mix, and NEB LongAmp Taq DNA Polymerase, were tested. Of these, the best PCR performance and good quality Nanopore sequence data were obtained with the Quantabio repliQa HiFi ToughMix polymerase (Figure 6A). The performance of Takara Bio PrimeSTAR reagent was inconsistent across samples (Figure 6C), and NEB Q5 and LongAmp reagents were found to be unsuitable for multiplex PCR amplification.

[0189] ONT library preparation:

[0190] For ONT (Oxford Nanopore Technologies) sequencing, the PCR-amplified DNA that showed positive results on the gel was selected for further processing. Before sequencing, both the pools of the respective samples were mixed together, and sequencing was performed using the Rapid Barcoding Kit SQK-RBK110.96, as per the manufacturer’s protocol. Sequencing was allowed to continue until a minimum depth of 50x was achieved for all the target amplicons. The real-time assessment of sequencing depth was facilitated using Rampart software.

[0191] LSDV variant identification and phylogenetic analysis

[0192] Raw sequence reads were base called and demultiplexed using the Guppy super-accurate algorithm, followed by quality control with fastp. High-quality reads were then mapped to the reference genome using minimap2, primer sequences were trimmed with Bamclipper, and variant calling was performed sequentially with medaka and longshot. Variants passing quality filters were used for reference-guided assembly with BCFtools and Bedtools, and annotation was carried out using snpEFF; a Python script was further used to generate nucleotide-level depth plots. For phylogenetic analysis, alignments were generated with MAFFT, maximum-likelihood trees were inferred with IQ-TREE, and both rooted and unrooted trees were visualized using iTOL. A bash script named as virAssem_2.sh was developed for automated analysis of sequence data.

[0193] Phylogenomics and evolution of LSDV

[0194] Phylogenetic analysis was carried out for all available LSDV genomes (41 from this study and 133 available from the NCBI GenBank database and GitHub repository). The sequences were aligned, and the IQTREE2 maximum likelihood algorithm was used to construct the phylogenetic tree. Phylogenetic analysis was performed using IQ-TREE with ModelFinder to select the best-fit substitution model. The K3Pu+F+I+R5 model was chosen as optimal according to the Bayesian Information Criterion (BIC).

[0195] It is apparent from the phylogeny that LSDV strains from India, sampled in 2022 and 2023, form two distinct clades. A small number of LSDV strains from India sampled in 2022 grouped within clade 1.2.2, which also includes the Neethling 2490 strain (NC_003027.1; used as reference for variant identification), which was originally isolated from Kenya in 1959, 2 Bangladesh strains sampled prior in 2021, and other strains from India sampled between 2016 and 2019 (Figure 7). The LSDV strain genomes reported from China, Thailand, and Vietnam between 2016 and 2022 formed a distant clade (clade 2.5) and are closer to viruses grouping in clade 1.1 (Figure 7).

[0196] While most of these variations can be seen in previously reported genomes, a number of novel mutations conserved across the LSDV genomes from field samples were detected. These include 10 non-synonymous, 5 synonymous and 19 indels (in coding regions). Indels in LSDV019, LSDV026 and LSDV087 genes disrupted the respective coding sequences, potentially resulting in loss of function. Nine commonly occurring mutations were enriched in the genes with viral virulence and host range function (LSDV005, LSDV019, LSDV067, LSDV128, LSDV144, LSDV150, LSDV151). The higher proportion of mutations in virulence and replication-associated genes may reflect selective pressures on LSDV to adapt to new hosts or environmental conditions. This pattern, also noted in previous Indian isolates, supports the possibility of ongoing diversification of viral sub-lineages.

[0197] Example 4

[0198] Kit for Detection

[0199] The kit comprises:

[0200] • NMDPP (outer and inner sets, 6 primers each) having SEQ ID Nos: 97-108

[0201] • Master mix (dNTPs, buffer, DNA polymerase)

[0202] • Positive control (synthetic DNA segments)

[0203] • Negative control (reaction mixture without nucleic acid)

[0204] • Nuclease-free water

[0205] Example 5

[0206] Kit for Amplification

[0207] The kit comprises:

[0208] • Pool of primers having SEQ ID Nos: 1-96

[0209] • Master mix (dNTPs, buffer, DNA polymerase)

[0210] • Positive control (synthetic DNA segments)

[0211] • Negative control (reaction mixture without nucleic acid)

[0212] • Nuclease-free water

[0213] ADVANTAGES OF THE INVENTION

[0214] 1. The present invention provides a non-invasive sample collection, detection and genotyping methods which can be helpful in the disease surveillance of LSDV in dairy animals.

[0215] 2. The present invention provides a robust and low-cost diagnostic method suitable for large- scale surveillance to detect and genotype the LSDV, followed by sequencing using Oxford Nanopore Technology (ONT).

[0216] 3. The multiplex primer panel of the present invention can amplify shorter genomic amplicons of size about 0.5 kb with uniform efficiency, useful for virus detection.

[0217] 4. The developed multiplex detection primer panel (LSDV_NMDPP) of the present invention provides robust detection of LSDV at concentrations as low as about 0.1 fg in a DNA sample, corresponding to detection of about 30 or more copies of the target genome, thereby demonstrating the high sensitivity of the nested multiplex PCR assay.

[0218] 5. The whole genome amplification primer panel LSDV_WGSPP_3.5 of the present invention can amplify 99.8% of the 151 kb LSDV genome in only 2 PCR reactions, which can be further pooled and sequenced as a single sample to get the complete genome sequence of LSDV for the purpose of genotyping and viral variant identification.

[0219] 6. The present invention provides primer panels for nested multiplexed PCR-based detection and whole genome amplification and sequencing that are useful for the analysis of field samples at low cost and provide results in a short time span.

Claims

We Claim:

1. A primer panel for genome amplification of Lumpy Skin Disease Virus (LSDV), said primer panel having pools of primers, each pool comprising:(i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33,34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81, 82,85, 86, 89, 90, 93, 94 or variants thereof; and(ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32,35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83,84, 87, 88, 91, 92, 95, 96 or variants thereof.

2. A composition for genome amplification of Lumpy Skin Disease Virus (LSDV), wherein said composition comprises the primer panel as claimed in claim 1 and optionally comprising a master mix containing dNTPs, buffer, and a DNA polymerase.

3. A primer set for detection of Lumpy Skin Disease Virus (LSDV) said primer set comprising:(i) set of outer primers having SEQ ID NOs: 97-102 or variants thereof; and(ii) set of inner primers having SEQ ID NOs: 103-108 or variants thereof.

4. A method for the detection of Lumpy Skin Disease Virus (LSDV) in a sample by nested multiplex PCR, the method comprising the steps of:(i) contacting denatured target DNA in the sample with a primer set comprising outer primers of SEQ ID NO: 97-102 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to produce a first PCR product; and(ii) contacting said first PCR product with a primer set comprising inner primers of SEQ ID NOs: 103-108 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to produce the amplified copies of nucleic acids are obtained.

5. The method as claimed in claim 4, wherein the method involves primary and secondary PCR cycles, the primary PCR cycle comprising:(i) initial denaturation at 70-98 °C for 3-7 minutes, followed by one or more cycles of 70-98 °C denaturation for 20-40 seconds;(ii) annealing at 40-70 °C for 20-40 seconds;(iii) extension at 65-80 °C for 1-3 minutes, followed by final extension at 65-80 °C for 2-5 minutes and; the secondary PCR cycle comprising:(i) initial denaturation at 70-98°C for 3-7 minutes, followed by one or more cycles of 70-98°C, preferably at 94°C denaturation for 20-40 seconds;(ii) annealing at 40-70°C, preferably at 55°C for 20-40 seconds; and(iii) extension at 65-80 °C for 1-2 minutes, followed by final extension with at 65-80 °C, preferably at 72 °C for 1-3 minutes.

6. A method for the whole genome sequencing of the Lumpy Skin Disease Virus (LSDV) by multiplex polymerase chain reaction (PCR), wherein the method comprises:(i) mixing half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81, 82, 85, 86,89, 90, 93,94 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product;(ii) mixing the other half of the sample containing the target DNA with a primer pool containing a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83, 84, 87, 88, 91, 92, 95, 96 or variants thereof, a master mix comprising deoxynucleotide triphosphates (dNTPs), a buffer, and a DNA polymerase to obtain the PCR product; and(iii) processing the PCR products thus obtained for library preparation for sequencing.

7. The method as claimed in claim 6, the multiplex PCR comprises:(i) denaturation at 90-99 °C for 20-40 seconds, followed by one or more cycles of 90-99 °C denaturation for 10-40 seconds, preferably, at 98 °C; and(ii) annealing and extension at 40-70 °C, preferably 65 °C.

8. The method as claimed in claim 6, wherein the amplicons in each of the PCR products have about 3.5 kb size having overlapping regions of about 0.5 kb size.

9. The method as claimed in claim 4 or 6, wherein the sample is a bovine sample, said bovine sample being a clinical sample selected from skin swab, nasal swab, blood, milk, and lesion swab or an environmental sample selected from drinking water, wastewater, soil, and feed.

10. A kit for the detection of Lumpy Skin Disease Virus (LSDV) comprising;(i) set of outer primers having SEQ ID NOs: 97-102 or variants thereof;(ii) a set of inner primers having SEQ ID NOs: 103-108 or variants thereof;11. A kit for the amplification of Lumpy Skin Disease Virus (LSDV) comprising:(i) a set of primers having SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33,34, 37, 38, 41, 42, 45, 46, 49, 50, 53, 54, 57, 58, 61, 62, 65, 66, 69, 70, 73, 74, 77, 78, 81, 82,85, 86, 89, 90, 93, 94 or variants thereof;(ii) a set of primers having SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32,35, 36, 39, 40, 43, 44, 47, 48, 51, 52, 55, 56, 59, 60, 63, 64, 67, 68, 71, 72, 75, 76, 79, 80, 83,84, 87, 88, 91, 92, 95, 96 or variants thereof.

12. The kit as claimed in claim 10 or 11, wherein the kit further comprises:(i) deoxynucleotide triphosphates (dNTPs);(ii) a buffer;(iii) DNA polymerase;(iv) optionally, a positive control containing synthetic DNA segments of amplification targets;(v) optionally a negative control containing a reaction mixture without any nucleic acid; and(vi) nuclease-free water.

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