摘要
目的分析DNA实时荧光恒温扩增法(简称"恒温扩增法")在结核病临床检测中的应用价值。方法收集2019年1—6月西安市胸科医院收治的疑似肺结核患者2421例,其中共有356例患者留取的痰液标本同时进行了BACTEC MGIT 960液体培养、利福平耐药实时荧光定量核酸检测(GeneXpert MTB/RIF法)、传统DNA实时荧光定量核酸检测(FQ-PCR法)、结核分枝杆菌RNA实时荧光恒温扩增(SAT-RNA法)、恒温扩增法检测。以MGIT 960技术检测结果为金标准,计算其他4种检测技术的敏感度、特异度、阳性预测值、阴性预测值、符合率、Kappa值。对恒温扩增法阳性的121份核酸样本使用熔解曲线法检测利福平ropB基因,研究恒温扩增法提取核酸应用于耐药基因检测的价值。结果以MGIT 960技术检测结果为金标准,恒温扩增法的敏感度(78.83%,108/137)低于GeneXpert MTB/RIF法(95.62%,131/137),高于FQ-PCR法(74.45%,102/137)和SAT-RNA法(59.12%,81/137),差异均有统计学意义(χ^(2)值分别为47.437、43.654、29.467,P值均<0.001)。GeneXpert MTB/RIF法的ROC曲线下面积最大(0.910),随后依次为恒温扩增法(0.860)、FQ-PCR法(0.854)、SAT-RNA法(0.789)。恒温扩增法阳性的核酸标本用熔解曲线法检测利福平ropB耐药基因,原核酸和稀释5倍后核酸荧光检测的dt值(探测时间,detection time,1 dt=1min)在2630时,ropB基因的检出率分别为93.55%(29/31)和90.32%(28/31);dt值在1525时,ropB基因的检出率分别为94.44%(34/36)和100.00%(36/36)。结论通过与各种检测技术比较,恒温扩增法具有临床应用价值,并适用于基层医院的结核病诊断。
Objective To analyze the application value of DNA real-time fluorescence isothermal amplification method in the clinical detection of tuberculosis.Methods A total of 2421 patients with suspected pulmonary tuberculosis in Xi’an Chest Hospital from January to June 2019 were checked.Sputum samples collected from 356 patients were simultaneously subjected to BACTEC MGIT 960 liquid culture,real-time fluorescent quantitative nucleic acid detection of rifampicin resistance(GeneXpert MTB/RIF method),traditional real-time fluorescent quantitative nucleic acid detection of DNA(FQ-PCR method),detection of M.tuberculosis RNA by real-time fluorescence constant temperature amplification(SAT-RNA method)and isothermal amplification method.Taking the MGIT 960 test results as the gold standard,we calculated the sensitivity,specificity,positive predictive value,negative predictive value,coincidence rate,and Kappavalue of the other four tests.The melting curve method was used to detect rifampin ropBgene in 121 nucleic acid samples positive for isothermal amplification method,to study the value of extracting nucleic acid with isothermal amplification method for drug resistance gene detection.Results Taking MGIT 960 test results as the gold standard,the sensitivity of isothermal amplification method(78.83%,108/137)was lower than GeneXpert MTB/RIF(95.62%,131/137),higher than FQ-PCR(74.45%,102/137)and SAT-RNA(59.12%,81/137),differences were statistically significant(χ^(2)=47.437,43.654,29.467;Pvalues were all<0.001).The area under curve of the ROC curve for GeneXpert MTB/RIF was the largest(0.910),followed by the isothermal amplification method(0.860),FQ-PCR(0.854)and SAT-RNA(0.789).Conducting melting curve test on isothermal amplificated nucleic acid samples,when the dt value(detection time,1 dt=1 min)of the original nucleic acid and the 5 times diluted nucleic acid was 26-30,the detection rates of the ropBgene were 93.55%(29/31)and 90.32%(28/31)respectively;when the dt value was15-25,the detection rates were 94.44%(34/36)and 100.00
作者
杨翰
李爱芳
王佩
党丽云
YANG Han;LI Ai-fang;WANG Pei;DANG Li-yun(Xi’an Chest Hospital,Xi’an 710100,China)
出处
《中国防痨杂志》
CAS
CSCD
2020年第12期1294-1298,共5页
Chinese Journal of Antituberculosis
基金
陕西省重点研发计划(2018SF-254)。
关键词
分枝杆菌
结核
核酸扩增技术
实验室技术和方法
对比研究
数据说明
统计
Mycobacterial tuberculosis
Nucleic acid amplification techniques
Laboratory techniques and procedures
Comparative study
Data interpretation,statistical
