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Development of a rapid loop-mediated isothermal amplification (LAMP) assay for visual detection of porcine parvovirus (PPV) and its application
Brazilian Journal of Microbiology ( IF 2.2 ) Pub Date : 2021-07-09 , DOI: 10.1007/s42770-021-00569-1
S Rajkhowa 1 , M Choudhury 1 , S R Pegu 1 , D K Sarma 2 , I Hussain 3
Affiliation  

Porcine parvovirus (PPV) infection is one of the most important causes of reproductive failure in pigs impacting the piggery industry globally with huge economic losses. A cost-effective, simple, rapid, specific, and sensitive method is critical for monitoring PPV infection on pig farms. The main aim of the present study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of porcine parvovirus (PPV) in pigs. A set of six LAMP primers including two outer primers, two inner primers, and two loop primers were designed utilizing the conserved region of capsid protein VP2 gene sequences of PPV and was applied for detection of PPV from porcine samples. Time and temperature conditions for amplification of PPV genes were optimized to be 30 min at 63 °C. The developed assay was ten-fold more sensitive than conventional PCR with analytical sensitivity of 20 pg and 200 pg, respectively. This is the first report of detection of PPV by LAMP assay from India. The assay did not cross-react with porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), or classical swine fever virus (CSFV). The LAMP assay was assembled into a LAMP assay kit of 20 reactions and was validated in different laboratories in India. The newly developed LAMP assay was proved to be a specific, sensitive, rapid, and simple method for visual detection of PPV which does not require even costly equipments for performing the test. It complements and extends previous methods for PPV detection and provides an alternative approach for detection of PPV.



中文翻译:

猪细小病毒(PPV)视觉检测快速环介导等温扩增(LAMP)法的开发及其应用

猪细小病毒 (PPV) 感染是导致猪繁殖失败的最重要原因之一,对全球养猪业造成巨大经济损失。一种具有成本效益、简单、快速、特异性和灵敏的方法对于监测猪场的 PPV 感染至关重要。本研究的主要目的是开发和评估一种用于快速目测猪细小病毒 (PPV) 的环介导等温扩增 (LAMP) 检测方法。利用PPV衣壳蛋白VP2基因序列的保守区设计了一套6条LAMP引物,包括2条外引物、2条内引物和2条环引物,用于猪样本中PPV的检测。PPV 基因扩增的时间和温度条件优化为 63 °C 下 30 分钟。所开发的检测方法的灵敏度比传统 PCR 高 10 倍,分析灵敏度分别为 20 pg 和 200 pg。这是印度首例通过 LAMP 法检测 PPV 的报告。该测定未与猪圆环病毒 2 型 (PCV2)、猪繁殖与呼吸综合征病毒 (PRRSV) 或经典猪瘟病毒 (CSFV) 发生交叉反应。LAMP 检测被组装成一个包含 20 个反应的 LAMP 检测试剂盒,并在印度的不同实验室进行了验证。新开发的 LAMP 检测被证明是一种特异性、灵敏、快速和简单的 PPV 视觉检测方法,它甚至不需要昂贵的设备来进行检测。它补充和扩展了以前的 PPV 检测方法,并提供了一种检测 PPV 的替代方法。

更新日期:2021-07-09
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