猪繁殖与呼吸综合征病毒TaqMan-MGB实时荧光定量RT-PCR方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2019.01.028

Abstract

Abstract:

In order to quickly and accurately detect porcine reproductive and respiratory syndrome (PRRSV),one TaqMan-MGB Real-time RT-PCR assay for detection of PRRSV was established using specific primers and probe based on PRRSV gene.The developed Real-time RT-PCR method could detect PRRSV classic strain,highly pathogenic mutant strain,as well as the new NADC30-like strain at the same time.The specificity,sensitivity and repeatability of the established method were detected.120 clinical samples were detected using the developed Real-time RT-PCR method and the conventional PCR method.The results showed that TaqMan-MGB Real-time RT-PCR method was specific for PRRSV classic strain,highly pathogenic mutant strain,as well as the NADC30-like strain,and the test results were negative with CSFV,PEDV,TGEV,RV,PRV,PPV and PCV2.The method showed a good linear relationship within the template ranges from 10¹ to 10⁸ copies/μL, and its sensitivity was 100 times that of routine PCR assay.The correlation of the standard curve was 0.9999,and the efficiency was 93%.The limit of detection concentration was 10¹ copies/μL.The coefficient of variation in the intra-and inter-assays were 0.17% to 0.90% and 0.65% to 2.34%,respectively.The positive detection rate of PRRSV in clinical samples using the developed Real-time RT-PCR method (59.2%) was higher than that using the conventional PCR method (44.2%).The establishment of Real-time RT-PCR method provided a rapid and accurate detection means for early diagnosis and epidemiological investigation of the disease.

Key words: porcine reproductive and respiratory syndrome virus (PRRSV); TaqMan-MGB Real-time RT-PCR assay; clinical samples

CLC Number:

S852.65⁺1

LI Xiaofei, CHEN Ting, WEI Xiaoxiao, SUN Airong, HUANG Yan, GE Xiaojie, XU Ting, WANG Caihong, QIN Liting. Establishment and Application of TaqMan-MGB Real-time RT-PCR Assay for Detection of Porcine Reproductive and Respiratory Syndrome Virus[J]. , 2019, 46(1): 239-246.

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Establishment and Application of TaqMan-MGB Real-time RT-PCR Assay for Detection of Porcine Reproductive and Respiratory Syndrome Virus

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