A real-time qPCR method for early detection of Bombyx mori Bidensovirus (BmBDV) infection in silkworm

Q4 Biochemistry, Genetics and Molecular Biology Animal Gene Pub Date : 2022-08-01 DOI:10.1016/j.angen.2022.200132

Gundi Raghavendar , Tania Gupta , A. Ramesha , Vankadara Sivaprasad , Kangayam M. Ponnuvel

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Abstract

Bombyx mori bidensovirus (BmBDV) replicates in the midgut cells of the silkworm to cause fatal flacherie disease. Flacherie disease causes a major impact in silk production and severe economic loss in the silk industries of India. BmBDV possesses two single stranded DNA that encodes for putative protein-primed DNA polymerase and adopts its own mechanism for replication. Development of rapid, sensitive and simple-to-use novel technology such as real-time polymerase chain reaction (RT-qPCR) will be useful in detection of BmBDV in silkworms. This RT-PCR assay will help to understand the significant pathology of BmBDV. The primers in specific to VD2 ORF1 region of BmBDV were designed to study their sensitivity and specificity. The detection limit of qPCR was found to be 3.01 × 10¹ copy number in the plasmid DNA with 96.3% efficiency in RT-PCR. In accordance, results shown in our study are 1000 times more sensitive than the conventional PCR with a detection limit of 3 × 10⁴ in PCR assay. Apart from displaying an increased sensitivity, these primers show high specificity towards BmBDV pathogenicity. On detection of flacherie infection studies with the specific primers shows gradual increase in detection of infectivity of BmBDV from 12 h to post infection for 48 h. In this case, the copy number reported at 48 h was found to be >10⁷ copies/reaction through qPCR analysis. The validation of this method was conducted using 90 diseased samples collected from 10 different sericulture farms. Here, detected virus copy numbers range from 1.24 × 10³ to 1.08 × 10¹⁰ where the optimization of RT-PCR could be used as a valuable tool in detection of BmBDV virus. Further aid in formulating appropriate disease control strategies, screening silkworm breeds for resistance during early infection stages are initiating reasons for this study. This particular analysis will provide a platform for developing silkworm hybrids to prevent further crop loss at the field level.

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家蚕Bidensovirus (BmBDV)感染的实时qPCR早期检测方法

家蚕病毒(Bombyx mori bidensovirus, BmBDV)在家蚕的中肠细胞中复制，引起致命的绒毛病。丝织病对印度的丝绸生产造成重大影响，并造成严重的经济损失。BmBDV具有两条单链DNA，编码推定的蛋白质引物DNA聚合酶，并采用自己的复制机制。实时聚合酶链反应(RT-qPCR)等快速、灵敏、简便的新技术将有助于家蚕BmBDV的检测。该RT-PCR检测将有助于了解BmBDV的重要病理。设计BmBDV VD2 ORF1区特异性引物，研究其敏感性和特异性。qPCR检测限为3.01 × 101拷贝数，RT-PCR检测效率为96.3%。因此，本研究结果的灵敏度是传统PCR的1000倍，PCR检测限为3 × 104。这些引物除了显示出更高的敏感性外，还显示出对BmBDV致病性的高特异性。用特异引物检测乳头状病毒感染研究表明，从感染后12小时到感染后48小时，BmBDV的检测传染性逐渐增加。在本例中，通过qPCR分析，48小时报告的拷贝数为107拷贝/反应。采用10个不同蚕桑养殖场采集的90份病样对该方法进行了验证。检测到的病毒拷贝数范围为1.24 × 103 ~ 1.08 × 1010，优化后的RT-PCR可作为检测BmBDV病毒的一种有价值的工具。进一步帮助制定适当的疾病控制策略，在早期感染阶段筛选家蚕品种的抗性是本研究的启动原因。这一特殊的分析将为开发蚕种杂交品种提供一个平台，以防止田间进一步的作物损失。

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Animal Gene Agricultural and Biological Sciences-Insect Science

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期刊介绍： Gene Reports publishes papers that focus on the regulation, expression, function and evolution of genes in all biological contexts, including all prokaryotic and eukaryotic organisms, as well as viruses. Gene Reports strives to be a very diverse journal and topics in all fields will be considered for publication. Although not limited to the following, some general topics include: DNA Organization, Replication & Evolution -Focus on genomic DNA (chromosomal organization, comparative genomics, DNA replication, DNA repair, mobile DNA, mitochondrial DNA, chloroplast DNA). Expression & Function - Focus on functional RNAs (microRNAs, tRNAs, rRNAs, mRNA splicing, alternative polyadenylation) Regulation - Focus on processes that mediate gene-read out (epigenetics, chromatin, histone code, transcription, translation, protein degradation). Cell Signaling - Focus on mechanisms that control information flow into the nucleus to control gene expression (kinase and phosphatase pathways controlled by extra-cellular ligands, Wnt, Notch, TGFbeta/BMPs, FGFs, IGFs etc.) Profiling of gene expression and genetic variation - Focus on high throughput approaches (e.g., DeepSeq, ChIP-Seq, Affymetrix microarrays, proteomics) that define gene regulatory circuitry, molecular pathways and protein/protein networks. Genetics - Focus on development in model organisms (e.g., mouse, frog, fruit fly, worm), human genetic variation, population genetics, as well as agricultural and veterinary genetics. Molecular Pathology & Regenerative Medicine - Focus on the deregulation of molecular processes in human diseases and mechanisms supporting regeneration of tissues through pluripotent or multipotent stem cells.