实时荧光PCR检测B型流感病毒方法的建立及病毒培养法验证

Establishment of FQ-PCR Method Detecting Influenza B Virus and Verification with Culturing Method

目的建立B型流感病毒实时荧光PCR检测(FQ-PCR)法,并与常规病毒培养法比较,判断二者结果的相关性,确定两种检测方法各自更适合的应用领域。方法建立FQ-PCR法并对76株经Quidel胶体金试纸条鉴定型别的流感病毒分离培养株进行检测,比较其敏感性和特异性。21～223梯度稀释的B型流感病毒分别用FQ-PCR法和病毒培养法进行检测,记录FQ-PCR的Ct值及培养物的血凝效价,并对FQ-PCR阳性但血凝无效价的梯度进行盲传,再次检测HA效价,比较二法的灵敏度及相关性。对临床采集的881份咽拭子标本做FQ-PCR检测,同时进行病毒培养并盲传、血凝试验及Quidel胶体金试纸条分型,综合比较两种检测方法之间的差异。结果在76株已知流感病毒标本中,FQ-PCR方法检出B型16份,与Quidel胶体金分型结果相符。FQ-PCR方法的灵敏度约高出常规培养法的25～26倍。FQ-PCR法对临床咽拭子的检测灵敏度高,无非特异性反应。结论流感病毒FQ-PCR法具有与组织培养法一致的特异性,前者操作简便快捷,灵敏度更高,在流感病毒临床快速检测和鉴定中具有更广阔的应用前景。

Objective To establish a fluorescent real-time polymerase chain reaction （FQ-PCR） method detecting influenza B virus, and compare with the routine culture method. Methods FQ-PCR was established to test 76 cases of which influenza infection and the virus subtype were confirmed by culture and agglutination methods. 2^1-2^23 fold diluted virus samples were used to evaluate the FQ-PCR and culture methods. For those samples showed positive in the FQ-PCR test but negative in agglutination test were performed a double blind test to evaluate the accuracy and sensitivity. 881 samples of pharyngeal swabs were tested by both FQ-PCR and culture method. Results For the 76 known virus sample, 16 were detected as B type by FQ-PCR and the result was matched with that by Quidel method. The FQ-PCR method is 2^5 -2^6 folds higher than the culture method and is a very sensitive for the pharyngeal swabs samples. Conclusion FQ- PCR is rapid, specific and sensitive method for the detection and typing of influenza B virus.