极速实时荧光聚合酶链反应与常规方法对新型布尼亚病毒检测的评价

目的探讨极速实时荧光聚合酶链反应(polymerase chain reaction,PCR)、实时荧光PCR、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和胶体金免疫层析法(gold immunochromatography assay,GICA)4种方法检测新型布尼亚病毒的特异度和灵敏度,为发热伴血小板减少综合征的早期诊断提供依据。方法采集2017年6月1日至9月30日山东大学附属济南市传染病医院86例临床诊断为发热伴血小板减少综合征患者的血清样本,分别应用极速实时荧光PCR、实时荧光PCR、ELISA和GICA 4种方法进行检测。统计学分析采用χ^2检验。结果86份患者血清标本中,极速实时荧光PCR、实时荧光PCR、IgM-ELISA、IgG-ELISA、IgM-GICA、IgG-GICA的新型布尼亚病毒阳性分别为82份(95.34%)、79份(91.86%)、41份(47.67%)、8份(9.3%)、19份(22.09%)和3份(3.49%)。极速实时荧光PCR特异度为100%,灵敏度达到1×103拷贝/mL,3次重复扩增试验显示其Ct值变异系数均<2%。在发热伴血小板减少综合征进展的1期、2期、3期病程中,极速实时荧光PCR的阳性检出率为41份(97.62%)、34份(94.44%)、7份(87.50%),实时荧光PCR的阳性检出率为39份(92.86%)、33份(91.67%)、7份(87.50%),在1期和2期两个病程,极速实时荧光PCR阳性检出率略高;IgM-ELISA阳性检出率从1期(28.57%)到3期(87.50%)显著增高,2期、3期与1期相比,差异均有统计学意义(χ^2=8.347、7.561,均P<0.01);IgM-GICA的阳性检出率从1期(14.29%)到2期(33.33%)也有增高,差异有统计学意义(χ^2=3.962,P<0.05),但与其他方法相比,其检出率偏低。1期,实时荧光PCR阳性检出率显著高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=33.740、55.080、49.010、64.340,均P<0.01)。2期,实时荧光PCR的阳性检出率高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=7.700、46.720、23.700、50.630,均P<0.01)。3期,极速实时荧光PCR、实时荧光PCR和IgM-ELISA表现出同样高的阳性检出率,远高于IgG-ELISA和GICA(IgM和IgG)。实时荧光PCR阳性检出率和IgG-ELISA、IgM-GICA、IgG-GICA之间差异均有统计学意义(均χ^2=6.250,P<0.05)。结论极速实时荧光PCR在新型布尼亚病毒的早期检测中有更高的灵敏度和特异度,且重复性好、稳定度高,与传统实时荧光PCR相比大大缩短了扩增时间,对发热伴血小板减少综合征的早期快速诊断具有重要价值。

Evaluation of a new ultrafast real-time fluorescence polymerase chain reaction and common assays in the detection of novel Bunya virus

Objective To investigate the specificity and sensitivity of four methods including ultrafastreal-time fluorescence polymerase chain reaction(PCR),real-time fluorescence(RT)-PCR,enzyme-linked immunosorbent assay(ELISA)and gold immunochromatography assay(GICA)for the detection of novel Bunya virus,so as to provide experimental basis for the early diagnosis of severe fever with thrombocytopenia syndrome(SFTS).Methods Serum samples from 86 clinically diagnosis SFTS patients admitted to the Jinan Infectious Diseases Hospital Affiliated to Shandoug University were tested by ultrafast real-time fluorescence PCR,RT-PCR,ELISA and GICA during June 1 to September 30,2017.Chi-square test was used for statistical analysis.Results Among 86 serum samples,the positive rate of novel Bunya virus of ultrafast real-time fluorescence PCR,RT-PCR,IgM-ELISA,IgG-ELISA,IgM-GICA and IgG-GICA were 82(95.34%),79(91.86%),41(47.67%),8(9.3%),19(22.09%)and 3(3.49%),respectively.The specificity of ultrafast real-time fluorescence PCR was 100%,and the sensitivity was 1×103 copies/mL.Repeated amplification test showed that the variation coefficient of the computed tomography value was<2%.During phases one,two and three,the positive rates of ultrafast real-time fluorescence PCR were 41(97.62%),34(94.44%)and 7(87.50%),and RT-PCR were 39(92.86%),33(91.67%)and 7(87.50%),respectively.During phases one and two,the positive rate of ultrafast real-time fluorescence PCR was slightly higher.The positive rate of anti-novel Bunya virus antibody(IgM)tested by ELISA had a significant increase from phase one(28.57%)to phase three(87.50%).There were statistical differences between phase two and phase,as well as between phase three and phase one(χ^2=8.347 and 7.561,respectively,both P<0.01).IgM-GICA also had an increase from phase one(14.29%)to phase two(33.33%)(χ^2=3.962,P<0.01),while it was still lower than the other tests.In phase one,the positive rate of RT-PCR was higher than those of ELISA(both IgM and IgG)and GICA(both IgM and IgG)(χ^2=33.740,55.080,49.010 and 64.340,respectively,all P<0.01).In phase two,the positive rate of RT-PCR was higher than those of ELISA(both IgM and IgG)and GICA(both IgM and IgG)(χ^2=7.700,46.720,23.700 and 50.630,respectively,all P<0.01).In phase three,the positive rates of ultrafast real-time fluorescence PCR,RT-PCR and IgM-ELISA were equivalent,which were all higher than those of IgG-ELISA and GICA(both IgM and IgG).The positive rates of RT-PCR and IgG-ELISA,IgM-GICA and IgG-GICA were significantly different(allχ^2=6.250,all P<0.05).Conclusion In the early detection of novel Bunya virus,ultrafast real-time fluorescence PCR has higher sensitivity,specificity,good repeatability and high stability,which greatly reduces the amplification time compared with the traditional RT-PCR,and is of great value in the early and rapid diagnosis of SFTS.