摘要
目的基于SYBR GreenⅠ荧光染料建立鉴定鸟分枝杆菌(Mycobacterium avium,MA)的荧光PCR熔解曲线法,为临床鉴定MA提供简单、准确性高的分子生物学检测方法。方法以MA的特异性插入序列IS1311为检测靶标设计引物,在60℃的退火温度下,建立鉴定MA的SYBR GreenⅠ荧光PCR熔解曲线方法,并对MA等21种分枝杆菌标准株和金黄色葡萄球菌等5种常见致病菌进行检测,评价方法的特异性、检测限;以hsp65、16S rDNA普通PCR扩增产物测序分析为“金标准”,鉴定200株分枝杆菌临床分离株,评估建立方法鉴定MA的准确性。结果SYBR GreenⅠ荧光PCR熔解曲线方法在30个循环内能准确鉴定出21种分枝杆菌标准株和5种致病菌中的MA,检测限为5.6×10-2 ng/μL。200株分枝杆菌临床分离株中,以PCR测序鉴定出16株MA和184株其他分枝杆菌,以SYBR GreenⅠ荧光PCR熔解曲线方法鉴定出15株MA和185株非MA;本研究建立的方法和测序方法对MA的鉴定率差异无统计学意义(P>0.05);两法符合率为99.5%(199/200),一致性较高(Kappa值>0.75,P<0.01);建立方法的敏感性为93.8%(15/16),特异性为100.0%(184/184),阳性预测值为100.0%(15/15),阴性预测值为99.5%(184/185)。结论以MA特异性重复序列IS1311为基础建立的SYBR GreenⅠ荧光PCR熔解曲线方法,具有较高的灵敏度和特异性,为MA的临床检测提供新的途径。
Objective To establish a PCR melting curve method for the identification of Mycobacterium avium(MA)based on SYBR GreenⅠfluorescent dye,and then provide a simple,accurate molecular biological method for the clinical identification of MA.Methods The primers were designed based on the specific insertion sequence IS1311 of MA as the detection target.At the annealing temperature of 60℃,a SYBR GreenⅠfluorescence PCR melting curve method was established for the identification of MA.The specificity and the detection limit of the established method were evaluated by detecting 21 reference strains of mycobacterium including MA and 5 common pathogens including Staphylococcus aureus.Using the sequencing of PCR products of hsp65 and 16S rDNA as the“gold standard”,200 clinical isolates of mycobacterium were identified,and then the accuracy of the established method for the identification of MA was evaluated.Results The established SYBR GreenⅠfluorescence PCR melting curve method were able to accurately identify MA in 21 reference strains of mycobacterium and 5 pathogenic bacteria within 30 cycles,and the detection limit was 5.6×10-2 ng/μL.Among 200 clinical isolates of mycobacterium,16 strains of MA and 184 strains of other mycobacteria were identified by PCR sequencing,while 15 strains of MA and 185 strains of non-MA were identified by the established SYBR GreenⅠfluorescence PCR melting curve method.There was no significant difference in the identification rate of MA between PCR sequencing and the established method in this study(P>0.05),and there were high coincidence rate(99.5%,199/200)and high consistency(Kappa value>0.75,P<0.01)between them.The sensitivity,specificity,positive predictive value and negative predictive value of the established method were 93.8%(15/16),100.0%(184/184),100.0%(15/15)and 99.5%(184/185),respectively.Conclusion The established SYBR GreenⅠfluorescence PCR melting curve method based on the specific repeat sequence IS1311 of MA has high sensitivity and specificity,which provides a new way for the clinical detection of MA.
作者
王赛赛
潘丽萍
姜广路
李自慧
贾红彦
孙琦
张宗德
刘洋
WANG Saisai;PAN Liping;JIANG Guanglu;LI Zihui;JIA Hongyan;SUN Qi;ZHANG Zongde;LIU Yang(Department of Epidemiology,Beijing Chest Hospital,Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Institute,Beijing 101149,China;Beijing Key Laboratory for Drug-resistant Tuberculosis Research,Beijing Chest Hospital,Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Institute,Beijing 101149,China;National Clinical Laboratory on Tuberculosis,Beijing Chest Hospital,Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Institute,Beijing 101149,China)
出处
《临床检验杂志》
CAS
2020年第4期251-257,共7页
Chinese Journal of Clinical Laboratory Science
基金
国家自然科学基金(81902024)
“十二五”国家科技重大专项(2015ZX10004801-003,2017ZX10201301-004)
北京市自然科学基金(7192038)
北京市医管局登峰人才项目(DFL20181601)
通州区运河人才计划(YH201807,YH201921)。
