Development of a single-step SYBR green-based real-time PCR assay for detection and quantification of lumpy skin disease virus in cattle

IF 1 Q4 GENETICS & HEREDITY

Gene Reports Pub Date : 2025-02-14 DOI:10.1016/j.genrep.2025.102172

Sanganagouda K. , Sabha Kounin , K. Nagaraja , Basavaraj Sajjanar , Amitha Rena Gomes , B.H. Pavithra , Shivaraj Murag , B.R. Sumathi , B.P. Shankar , B.P. Shivashankar , H.C. Indresh , K.R. Anjan Kumar , Raveendra Hegade , D. Rathnamma

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引用次数: 0

Abstract

Background

Lumpy skin disease (LSD) is a vector-borne viral disease of cattle and water buffalos. The disease causes substantial economic losses in cattle across the Indian subcontinent. Effective diagnosis and control are critical for managing this disease. The primary aim of the current study is to develop a single-step SYBR green-based real-time PCR assay for the detection and quantification of Lumpy Skin Disease Virus (LSDV) in clinical samples.

Methods

A total of 160 samples were collected and subsequently viral propagation was carried out in Madin Darby Bovine Kidney (MDBK) cell lines. Initial viral identification was performed via conventional PCR employing newly synthesized primers, which targeted both the envelope protein (P32) and the G protein-coupled chemokine receptor (GPCR) gene. For the quantification of LSDV within infected nodular tissues, two distinct real-time PCR assays (Assay-I and Assay-II) were implemented. These assays utilized standard curves generated from specific GPCR amplicons of 610 bp and 786 bp, enabling precise absolute quantification. This methodological enhancement greatly improves the accuracy of LSDV prevalence assessment and supports more effective disease management strategies.

Results

LSDV viral loads in infected nodular tissues were measured using Real-Time PCR Assay-I and Assay-II, with average log mean values of 7.36 ± 0.18 and 7.27 ± 0.17 (n = 37), respectively. The lower detection limits were 284 and 153 copies per microliter (μl), with corresponding threshold cycle values of 25.75 ± 0.27 and 32.10 ± 0.64. Negative controls showed Ct values of 33.01 ± 0.37 and 33.39 ± 1.37 (n = 6), respectively. Additionally, LSDV was isolated in MDBK cell lines, demonstrating that primary cells can be effectively replaced with MDBK cell lines for viral isolation.

Conclusions

The single-step SYBR Green-based real-time PCR assay targeting the GPCR gene proved to be highly sensitive, specific, and reproducible for the detection and quantification of LSDV, offering a robust tool for monitoring and managing LSD.

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牛肿块性皮肤病病毒单步荧光定量检测方法的建立

背景肿块性皮肤病（LSD）是牛和水牛的一种媒介传播的病毒性疾病。这种疾病给整个印度次大陆的牛群造成了巨大的经济损失。有效的诊断和控制对于控制这种疾病至关重要。本研究的主要目的是开发一种基于SYBR绿色的单步实时PCR检测方法，用于临床样本中肿块性皮肤病病毒（LSDV）的检测和定量。方法采集160份样本，在马丁达比牛肾（MDBK）细胞系中进行病毒增殖。利用新合成的引物对病毒包膜蛋白（P32）和G蛋白偶联趋化因子受体（GPCR）基因进行常规PCR鉴定。为了定量感染结节组织中的LSDV，采用了两种不同的实时PCR方法（Assay-I和Assay-II）。这些实验利用610 bp和786 bp特异性GPCR扩增产生的标准曲线，实现了精确的绝对定量。这种方法的改进大大提高了LSDV患病率评估的准确性，并支持更有效的疾病管理策略。结果Real-Time PCR assayi和assayii检测感染结节组织中slsdv病毒载量，对数平均值分别为7.36±0.18和7.27±0.17 （n = 37）。下检出限分别为284和153拷贝/微升，相应的阈值循环值分别为25.75±0.27和32.10±0.64。阴性对照的Ct值分别为33.01±0.37和33.39±1.37 （n = 6）。此外，在MDBK细胞系中分离出LSDV，表明MDBK细胞系可以有效地取代原代细胞进行病毒分离。结论基于SYBR green的GPCR基因单步实时PCR检测和定量LSDV具有较高的灵敏度、特异性和可重复性，为LSD的监测和管理提供了可靠的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Gene Reports Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

3.30

自引率

7.70%

发文量

246

审稿时长

49 days

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.