直接免疫荧光法和实时荧光定量PCR法检测汉坦病毒效果的比较

Gomparison of direct immune-fluorescent assay and real-time quantitative PCR in detecting theHantavirus

目的比较直接免疫荧光法（DFA）和实时荧光定量PCR法检测鼠肺汉坦病毒（HV）带毒效果的差异。方法于2012年4-10月在陕西省户县、泾阳县和眉县3个肾综合征出血热（HFRS）高发县的居民区和野外鼠类活动地，采用鼠夹法捕获野鼠和家鼠共479只。解剖取鼠肺，1份鼠肺经冰冻切片后免疫荧光染色检测HV抗原，另1份经抽提组织RNA后应用实时荧光定量PCR法检测HV核酸。比较2种方法的病毒阳性检出率和检测结果的一致性。结果捕获的479只鼠中，包括家鼠105只、野鼠374只。DFA和实时荧光定量PCR两种方法在家鼠肺中均未检出HV，而DFA法检测野鼠带毒率为13．1％（49／374），实时荧光定量PCR法检测野鼠带毒率为14．7％（55／374），差异无统计学意义（X^2=0．402，P=0．526）。对每份鼠肺标本分别检测，DFA法检测鼠肺HV带毒率为10．2％（49／479），实时荧光定量PCR法检测带毒率为11．5％（55／479），两种方法比较差异无统计学意义（X^2=1．286，P=0．257），检测结果Kappa一致性系数（K）为68．2％，两种方法结果有高度一致性（X^2=11．759，P〈0．05）。以DFA法为标准，实时荧光定量PCR法在检测鼠肺HV的灵敏度为77．6％（38／49），特异度为96．1％（413／430）。DFA检测的9个疑似阳性结果中6个被实时荧光定量PCR方法证实，3个被否定。结论与DFA法相比，实时荧光定量PCR法同样可用于检测鼠肺中HV带毒率，而且结果更稳定。

Objective To compare the differences between the direct immuno-fluorescent assay （DFA） and real-time quantitative PCR in detecting the Hantavirus （HV） in rat lungs. Methods From April to October in 2012, a total of 479 rats were caught by mouse-trap in residential or wild areas in Huxian, Jingyang, and Meixian of Shaanxi province, where haemorrhagic fever with renal syndrome （HFRS） was highly prevalent. The rats were dissected to take the two lungs, one was frozen and applied immuno-fluorescent assay to detect HV antigen while the other one was extracted its RNA and detected HV nucleic acid by real-time quantitative PCR. Then we compared the positive rate of the two methods. Results Out of the 479 rats, 105 were caught from residential areas and the other 374 were caught from wild areas. Among the 105 rats caught from residential areas, no HV were detected out neither by DFA nor by real-time quantitative PCR. Among the 374 wild rats, 13.1% （49/374） were detected HV positive by DFA and 14. 7% （55/374） were detected HV positive by real-time quantitative PCR. The difference showed no statistical significance （ X^2 = 0. 402,P = 0. 526）. When detecting each lung sample,the HV positive rate was 10. 2% （49/479） under the detection by DFA while the HV positive rate was 11.5% （55/479） under thedetection by real-time quantitative PCR. The difference had no statistical significance （ X^2 = 1. 286, P = 0. 257） and the consistency coefficient was 68.2% under the paired chi-square test analysis, which showed high consistency （ u = 11. 759, P 〈 0. 05 ）. The sensitivity of real-time quantitative PCR to detect HV was 77.6% （38/49） comparing with DFA as standard, and the specificity was 96. 1% （413/430）. Out of the 9 suspected HV positive sample detected by DFA,6 were confirmed positive by real-time quantitative PCR and 3 were denied. Conclusion Compared with the DFA, real-time quantitative PCR could also be used to detect the infection of HV in rats, and the result might be more stable.