检测猪繁殖与呼吸综合征病毒和猪圆环病毒的二重荧光LAMP方法的建立

Development of Duplex Fluorescence-based Reverse Transcript Loop-mediated Isothermal Amplification Assay for Visible Detection of PRRSV and PCV

关键词:  猪繁殖与呼吸综合症病毒  猪圆环病毒  二重LAMP  检测

Key words:  porcine reproductive and respiratory syndrome viruses(PRRSV)  porcine circovirus 2 (PCV2)  duplex LAMP  detection

摘要:

建立一种二重荧光LAMP的检测猪繁殖与呼吸综合症病毒 (PRRSV)和猪圆环病毒（PCV）的快速诊断技术。根据已发表的PRRSV NSP2基因和PCV2 ORF2基因序列保守区域，分别设计并合成了两组针对PRRSV和PCV2序列的特异性引物和两条探针，两条探针分别标记不同的荧光基团，PRRSV-Probe 5’端标记FAM荧光基团，3’端标记BHQ1淬灭基团，PCV-Probe 5’端标记CY5.5荧光基团，3’端标记BHQ2淬灭基团。对组成反应体系中的引物、探针以及反应试剂进行优化，建立了二重荧光LAMP检测PRRSV和PCV2方法。对建立的方法进行特异性和敏感性测试，并用模拟混合样品和临床样品进行检测验证。结果显示，本研究建立的方法可以鉴别区分PRRSV和PCV2，特异性试验表明该方法只检测出PRRSV或PCV2，且与参试的对照病毒无交叉反应，特异性好。敏感性测试表明该方法最低检测PRRSV或PCV为100拷贝·mL-1，敏感性好。对临床样品的检测，结果与荧光定量PCR方法检测结果一致。本研究建立的PRRSV和PCV2二重荧光LAMP检测方法，具有良好的特异性和敏感性，可用于临床样品中检测PRRSV和PCV2。

Abstract:

The aim of this study was to establish a rapid nucleic acid visible detection assay for porcine reproductive and respiratory syndrome viruses(PRRSV) and porcine circovirus 2(PCV 2). The primers and probes targeting the PRRSV NSP2 gene and PCV 2 ORF2 gene were designed. The PRRSV-probe was labeled with FAM fluorescence-based at 5’terminal and BHQ1 quenches-base at 3’terminal.The PCV-probe was labeled with CY5.5 fluorescence-based at 5’terminal and BHQ2 quenches-base at 3’terminal.A duplex fluorescence-based loop-mediated isothermal amplification (LAMP) assay was developed. The specificity, sensitivity and stability?were evaluated using primers and probes of PRRSV and PCV 2, followed by clinical specimen verification. As results showed that the specificity of this assay was high, and there be able to detect PRRSV and PCV 2 without any cross-reaction with other known non-targeted bovine pathogens.The limits of detection of in assay were 100 copies. Different concentration template of PRRSV and PCV 2 could be identified when mixed together without any interference. Further,the results of this LAMP were 100% agreement with real-time PCR as a contrast in detection of clinical samples. In conclusion, a duplex LAMP assay for PRRSV and PCV was established and proved to be specific and sensitive. So this test method can be used for clinical identification for PRRSV and PCV 2.