重组酶聚合酶扩增技术检测结核分枝杆菌
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摘要
目的利用重组酶聚合酶扩增(Recombinase Polymerase Amplification,RPA)技术检测结核分支杆菌。方法采用Axxin T8-ISO扩增仪(TwistDX公司),反应温度设置为39℃,反应时间20min。检测分为实验组、阳性对照组和空白对照组。实验组模板DNA从痰液中提取。阳性对照模板DNA为H37Rv标准菌株样本DNA及卡介苗提取的DNA。空白对照为蒸馏水。结果 RPA技术可在20min内明显区分103～106 copies/mL的阳性质粒与阴性对照。对分离培养的结核杆菌(H37Rv)DNA及卡介苗提取的DNA,阳性克隆质粒进行等温扩增,结果结核杆菌分离株DNA、卡介苗DNA、阳性克隆质粒均有阳性扩增反应,而非结核分枝杆菌分离株和阴性对照无阳性扩增反应。灵敏度为100%,特异性为82%。结论 RPA技术利用了重组酶、单链结合蛋白、DNA聚合酶代替了传统PCR变性、退火、延伸的热循环过程,在常温25℃～42℃、15～30min内即可实现痕量核酸的快速扩增,可应用于快速检测人结核分枝杆菌,是一种全新的检测方法。
Objective To detect Mycobacterium tuberculosis by using Recombinase Polymerase Amplification(RPA).Methods The Axxin T8-ISO instrument(TwistDx)was used;the reaction temperature was set to 39℃,with the reaction time of 20 minutes.The test was conducted in 3 groups including the Experiment group,Positive control group and Blank control group.The template DNA of the experiment group was extracted from the sputum;while that of the positive control group was extracted from H37 Rv standard strain and BCG vaccine.The blank control was distilled water.Results A positive plasmid of 103-106 copies/mL was clearly distinguished from the negative control within 20 minutes.The positive clone plasmid was subjected to isothermal amplification on the isolated cultured TB(H37 Rv)DNA and the DNA extracted from BCG.The results showed that the Mycobacteriumtuberculosisisolate DNA,BCG DNA and positive clone plasmids had positive amplification reactions,while the non-M.tuberculosisisolates and negative controls did not.This test showed a sensitivity of 100% and a specificity of 82%.Conclusion RPA utilizes a recombinase,a single-stranded binding protein and a strand-displacing DNA polymerase to replace the thermal cycling process of denaturation,annealing and extension of conventional PCR,which,as a new detection method,can be used in the detection of human Mycobacterium tuberculosis to achieve rapid amplification of trace nucleic acids within 15-30 minutes at room temperature(25℃-42℃).
Objective To detect Mycobacterium tuberculosis by using Recombinase Polymerase Amplification(RPA).Methods The Axxin T8-ISO instrument(TwistDx)was used;the reaction temperature was set to 39℃,with the reaction time of 20 minutes.The test was conducted in 3 groups including the Experiment group,Positive control group and Blank control group.The template DNA of the experiment group was extracted from the sputum;while that of the positive control group was extracted from H37 Rv standard strain and BCG vaccine.The blank control was distilled water.Results A positive plasmid of 103-106 copies/mL was clearly distinguished from the negative control within 20 minutes.The positive clone plasmid was subjected to isothermal amplification on the isolated cultured TB(H37 Rv)DNA and the DNA extracted from BCG.The results showed that the Mycobacteriumtuberculosisisolate DNA,BCG DNA and positive clone plasmids had positive amplification reactions,while the non-M.tuberculosisisolates and negative controls did not.This test showed a sensitivity of 100% and a specificity of 82%.Conclusion RPA utilizes a recombinase,a single-stranded binding protein and a strand-displacing DNA polymerase to replace the thermal cycling process of denaturation,annealing and extension of conventional PCR,which,as a new detection method,can be used in the detection of human Mycobacterium tuberculosis to achieve rapid amplification of trace nucleic acids within 15-30 minutes at room temperature(25℃-42℃).
引文
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[7]Euler M,Wang Y,Nentwich O,et al.Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus[J].J Clin Virol,2012,54(4):308-312.
[8]Mekuria TA,Zhang S,Eastwell KC.Rapid and sensitive detection of Little cherry virus 2using isothermal reverse transcription-recombinase polymerase amplification[J].J Virol Methods,2014,205:24-30.
[9]Xia X,Yu Y,Weidmann M,et al.Rapid detection of shrimp white spot syndrome virus by real time,isothermal recombinase polymerase amplification assay[J].PLoS One,2014,9(8):e104667-e104667.
[10]Kersting S,Rausch V,Bier FF,et al.Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens[J].Microchimica Acta,2014,181(13/14):1715-1723.
[11]Euler M,Wang Y,Heidenreich D,et al.Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents[J].J Clin Microbiol,2013,51(4):1110-1117.
[12]Kr7lov K,Frolova J,Tudoran O,et al.Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples[J].J Mol Diagn,2014,16(1):127-135.收稿日期修回日期本文编辑
[2]Boyle DS,McNerney R,Teng LH,et al.Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification[J].PLoS One,2014,9(8):e103091.
[3]Meerak J,Wanichwecharungruang SP,Palaga T.Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85Bby incorporation of an autophagy inducing system[J].Vaccine,2013,31(5):784-790.
[4]DENG Guoying,YANG Shufeng,LIU Xin,et al.Progress in research on vaccines against Mycobacterium tuberculosis[J].Chin J Microecol,2018,(1):109-113.(in Chinese)邓国英,杨淑凤,刘欣,等.抗结核分枝杆菌疫苗的研究进展[J].中国微生态学杂志,2018,(1):109-113.
[5]Kersting S,Rausch V,Bier FF,et al.Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis[J].Malaria J,2014,13(1):99.
[6]Crannell ZA,Rohrman B,Richards-Kortum R.Equipment-free incubation of recombinase polymerase amplification reactions using body heat[J].PLoS One,2014,9(11):e112146.
[7]Euler M,Wang Y,Nentwich O,et al.Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus[J].J Clin Virol,2012,54(4):308-312.
[8]Mekuria TA,Zhang S,Eastwell KC.Rapid and sensitive detection of Little cherry virus 2using isothermal reverse transcription-recombinase polymerase amplification[J].J Virol Methods,2014,205:24-30.
[9]Xia X,Yu Y,Weidmann M,et al.Rapid detection of shrimp white spot syndrome virus by real time,isothermal recombinase polymerase amplification assay[J].PLoS One,2014,9(8):e104667-e104667.
[10]Kersting S,Rausch V,Bier FF,et al.Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens[J].Microchimica Acta,2014,181(13/14):1715-1723.
[11]Euler M,Wang Y,Heidenreich D,et al.Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents[J].J Clin Microbiol,2013,51(4):1110-1117.
[12]Kr7lov K,Frolova J,Tudoran O,et al.Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples[J].J Mol Diagn,2014,16(1):127-135.收稿日期修回日期本文编辑