猪繁殖与呼吸综合征病毒的逆转录重组酶介导等温扩增荧光检测方法的建立及应用

Establishment and application of a fluorescent assay for reverse transcription recombinase-mediated isothermal amplification of porcine reproductive and respiratory syndrome virus

摘要: 【目的】 开发研究一种临床上快速、简单和准确的检测PRRSV的方法。 【方法】 该研究建立一种基于逆转录重组酶介导等温扩增荧光检测PRRSV的方法。该方法通过针对PRRSV ORF4基因序列设计特异性引物和探针。 【结果】 荧光RT-RAA检测可在41℃下用引物和探扩增20 min,通过荧光定量PCR仪实时观察结果。该方法具有较高的灵敏度和较强的特异性,与其他猪病原体无交叉反应。 【结论】 使用荧光RT-RAA和PCR检测180份样品,两种方法之间的符合率98.3%。

Abstract: 【Objective】 There is a need for the development of a clinically rapid, simple, and accurate method for the detection of PRRSV. 【Methods】 This study aims to establish a method based on reverse transcription recombinase-mediated isothermal amplification fluorescence detection of PRRSV. The method was developed by designing specific primers and probes against the PRRSV ORF4 gene sequence. 【Results】 The fluorescent RT-RAA assay could be amplified with primers and probes at 41℃ for 20 min, and the results could be observed in real time by a fluorescence quantitative PCR instrument. The method had high sensitivity and strong specificity, and no cross-reactivity with other porcine pathogens. 【Conclusion】 The fluorescent RT-RAA and PCR used to test 180 samples showed the compliance rate between the two methods was 98.3%.