实时荧光核酸恒温扩增检测技术在结核病诊断中的临床应用

Evaluation of the simultaneous amplification and testing for diagnosis of Mycobacterium tuberculosis

目的 评估实时荧光核酸恒温扩增检测技术（simultaneous amplification and testing,SAT）在结核病患者的痰液、体液及病灶组织等标本中检测结核分枝杆菌的价值.方法 收集在广州市胸科医院就诊的疑似结核病患者的痰液734份、体液标本94份（包括68份胸腔积液、21份脑脊液、5份关节积液,以下统称“体液”）及病灶组织标本76份,共计904份标本.同时采用SAT法、改良罗氏培养法进行检测,培养阳性者进行基因芯片菌种鉴定.SAT法与改良罗氏培养法结果不相符的标本,用结核分枝杆菌聚合酶链（polymerase chain reaction technology for Mycobacterium tuberculosis,PCR-TB）荧光诊断试剂盒进行检测.采用x2检验比较SAT法与改良罗氏培养法对结核病患者的阳性检出率.结果 以改良罗氏培养结果为金标准,则SAT法在痰液、体液、病灶组织标本的敏感度、特异度、符合率分别为90.20％（230/255）、92.48％（443/479）、91.69％（673/734）;94.74％（18/19）、81.33％（61/75）、84.04％（79/94）;100.00％（14/14）、77.42％（48/62）、81.58％（62/76）.以扩大金标准（培养＋PCR法）比对,则SAT法在痰液、体液、病灶组织标本的敏感度、特异度、符合率分别为96.30％（260/270）、99.35％（461/464）、98.23％（721/734）;96.97％（32/33）、100.00％（61/61）、98.94％（93/94）;100.00％（27/27）、97.96％（48/49）、98.68％（75/76）.改良罗氏培养法和SAT法在痰液、体液、病灶组织标本的阳性检出率分别为34.74％（255/734）和36.24％（266/734）、20.21％（19/94）和34.04％（32/94）、18.42％（14/76）和36.84％（28/76）;痰标本两者的阳性率比较,差异没有统计学意义（x2=0.36,P＞0.05）.体液与病灶组织标本中,SAT法较改良罗氏培养法的阳性率高出13.83％和18.42％,差异有统计学意义（x2值分别为4.55、6.44,P值均＜0.05）.结论 SAT法检测痰液、体液及病灶组织标本中的结核分枝杆菌,具有快速、敏感度和特异度较高的优点,可提高结核分枝杆菌的检出率.

Objective To assess the clinical value of the isothermal RNA amplification assay （SAT） for detection of Mycobacterium tuberculosis in sputum,body fluid and nidus samples.Methods Seven hundred and thirtyfour sputum samples,94 samples of body fluid （including 68 thoracic effusion,21 cerebrospinal fluid and 5 joint effusion） and 76 nidus samples collected from suspected tuberculosis patients were tested by both SAT and L-J culture.The positive isolates were identified by Gene-chip method.The samples with different results between SAT and L-J culture were tested by Mycobacterium tuberculosis PCR （PCR-TB） fluorescence diagnosis kits.The detection rates of SAT and L-J culture were analyzed by Chi-square test.Results With the result of L-J culture as the reference,the sensitivity,the specificity and the coincidence rate of the sputum samples were 90.20％ （30/255）,92.48％ （443/479） and 91.69％ （673/734）,those of the fluid samples were 94.74％ （18/19）,81.33％ （61/75） and 84.04％ （79/94）,and those of the nidus samples were 100.00％ （14/14）,77.42％ （48/62） and 81.58％ （62/76）.With the result of PCR-TB as the reference,the sensitivity,the specificity and the accordance rate of the sputum samples were 96.30％ （260/270）,99.35％ （461/464） and 98.23％ （721/734）,those of the fluid samples were 96.97％ （32/33）,100.00％ （61/61） and 98.94％ （93/94）,and those of the nidus samples were 100.00％ （27/27）,97.96％ （48/49） and 98.68％ （75/76）.The detection rates the three different kinds of specimens by L-J culture and SAT were 34.74％ （255/734） vs 36.24％ （266/734）,20.21％ （19/94） vs 34.04％ （32/94）,18.42％ （14/76） vs 36.84％（28/76）.There was no difference in sputum samples between SAT and L-J culture （x2=0.36,P＞0.05）.However,there were significant differences in body fluids and nidus samples between SAT and L-J culture （x2=4.55 and 6.44,all P＜0.05）.Conclusion SAT is a rapid,sensitive and specific method for detection of Mycobacterium tuberculosis in kinds of clinical samples.