基于双分子信标对猪圆环病毒Ⅱ型的简单快速检测

Rapid Detection of Porcine Circovirus 2 Based on Double Molecular Beacons

根据猪圆环病毒（PCV2）基因组为单链DNA的特点,设计了两条可与PCV2基因组序列特异性杂交的分子信标,建立了基于双分子信标法检测PCV2的方法。实验结果表明,双分子信标法比单分子信标法的灵敏度更高。在10 mmol/L MgCl2、20 mmol/L Tris-HCl（pH=8.0）的杂交缓冲溶液,40℃孵育30 min的优化检测体系下,此方法可实现对检测样本在2~200 nmol/L线性范围内的检测,检出限可达1 nmol/L。将双分子信标检测体系用于18例可疑猪瘟样品病毒检测,其中8例呈PCV2阳性,检测结果与PCR法一致,证明此双分子信标法可用于实际猪感染PCV2的诊断。

A double-molecular beacons （DMB） based assay was developed for porcine circovirus 2（PCV2） detection. Two single-stranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences. The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon. Under the optimal conditions of 10 mmol/L MgCl2 , 20 mmol/L Tris-HCl （pH=8. 0）, 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L. The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method.