肠道轮状病毒巢式PCR检测方法的建立及初步应用

Establishment and Preliminary Application of Intestinal Rotavirus (RV) Nested PCR Detection Method

目的建立一种特异性和灵敏度高的检测婴幼儿腹泻粪便标本中肠道轮状病毒(RV)的巢式PCR方法,并探讨其在临床诊断中的初步应用价值.方法根据RV保守区基因序列并利用Primer5.0软件设计巢式PCR两对引物,建立一种特异性和灵敏度高的检测RV的巢式PCR方法,并评价其特异度和敏感度.同时采用所建立的巢式PCR法对136份临床样品进行检测,并用单一的RT-PCR法、胶体金和基因序列法进行结果验证.结果巢式PCR法只扩增检测出192 bp的RV特异性条带,对其他肠道病毒均呈阴性反应,特异性好;巢式PCR法最低能检测到8.705 fg/μL的RV;巢式PCR法与单一的RT-PCR法检测结果符合率为100%,但灵敏度低于胶体金法,差异有统计学意义(χ^2=2.615,P<0.05);将巢式PCR法扩增的RV产物进行核苷酸正反向测序,经BLAST比对,RV与EU679386、DQ870492、AF531912等20多株已知序列的同源性为98%~100%.结论建立了检测肠道病毒RV的巢式PCR法,具有特异性强、灵敏度高,且检测快速,值得临床推广应用.

Objective To establish a high specificity and sensitivity of detection of infant diarrhea stool specimens in the gut of rotavirus(RV)nested polymerase chain reaction(PCR),and to explore its preliminary application in clinical diagnosis.Methods according to the RV conservative district gene sequences and USES Primer5.0 software design two pairs of primers of nested PCR to establish a high specificity and sensitivity of detection of RV nested polymerase chain reaction(PCR),and to evaluate its specific degrees and sensitivity.136 clinical samples were tested with the established nested PCR method,and the test results were verified by single rt-pcr,colloidal gold and gene sequence analysis.Results the RV specific bands of 192bp were detected by nested PCR,and the other enteroviruses were negative and specific.The RV of 8.705fg/ul can be detected at least by nested PCR method.The detection results of nested PCR and single rt-pcr were 100%,but lowerthan the colloidal gold method,and the difference was statistically significant(χ^2=2.615,P<0.05).The RV products amplified by the nested PCR method were sequenced by nucleotide.By BLAST comparison,the homology of more than 20 known sequences,such as RV and EU679386,DQ870492 and AF531912,was 98%~100%.Conclusion the detection method of nested PCR for intestinal virus(RV)was established,which was characterized by high specificity,high sensitivity and rapid,and worthy of clinical application.