中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
Chinese Journal of Preventive Medicine
2015年
11期
1011-1015
,共5页
关丽%许松涛%聂凯%张丹%李鑫娜%许文波%马学军
關麗%許鬆濤%聶凱%張丹%李鑫娜%許文波%馬學軍
관려%허송도%섭개%장단%리흠나%허문파%마학군
聚合酶链反应%柯萨奇病毒感染%环介导逆转录等温扩增
聚閤酶鏈反應%柯薩奇病毒感染%環介導逆轉錄等溫擴增
취합매련반응%가살기병독감염%배개도역전록등온확증
Polymerase chain reaction%Coxsackievirus infections%Loop-mediated isothermal amplification
目的 建立基于羟基萘酚蓝(hydroxy naphthol blue, HNB)颜色变化的简单、灵敏和快速的环介导逆转录等温扩增技术(RT-LAMP)检测方法,应用于柯萨奇病毒A6型(coxsackievirus A6,CV-A6)的检测.方法 针对CV-A6的VP1基因设计6条特异引物,在等温条件下(63℃)进行50 min扩增反应.扩增前在反应体系中加入HNB,通过观察颜色变化进行检测结果判定.使用多种肠道病毒进行特异性验证,使用梯度稀释的体外转录CV-A6全VP1基因RNA进行灵敏度分析,同时与实时荧光定量逆转录PCR(rRT-PCR)检测结果进行比较,并对92份手足口病患者临床标本进行检测.结果 本研究建立的RT-LAMP方法对除CV-A6外的23种肠道病毒的检测结果均为阴性,灵敏度为100拷贝/反应,与rRT-PCR方法相当.在对92份手足口病临床标本的检测中,检测结果与rRT-PCR方法相符,Kappa值为1,灵敏度和特异性均为100%.结论 本研究建立的针对CV-A6的LAMP检测方法,特异度高,灵敏度与rRT-PCR相当,有望应用于CV-A6感染的快速筛选,具有在基层医疗卫生机构和现场推广与应用的潜力.
目的 建立基于羥基萘酚藍(hydroxy naphthol blue, HNB)顏色變化的簡單、靈敏和快速的環介導逆轉錄等溫擴增技術(RT-LAMP)檢測方法,應用于柯薩奇病毒A6型(coxsackievirus A6,CV-A6)的檢測.方法 針對CV-A6的VP1基因設計6條特異引物,在等溫條件下(63℃)進行50 min擴增反應.擴增前在反應體繫中加入HNB,通過觀察顏色變化進行檢測結果判定.使用多種腸道病毒進行特異性驗證,使用梯度稀釋的體外轉錄CV-A6全VP1基因RNA進行靈敏度分析,同時與實時熒光定量逆轉錄PCR(rRT-PCR)檢測結果進行比較,併對92份手足口病患者臨床標本進行檢測.結果 本研究建立的RT-LAMP方法對除CV-A6外的23種腸道病毒的檢測結果均為陰性,靈敏度為100拷貝/反應,與rRT-PCR方法相噹.在對92份手足口病臨床標本的檢測中,檢測結果與rRT-PCR方法相符,Kappa值為1,靈敏度和特異性均為100%.結論 本研究建立的針對CV-A6的LAMP檢測方法,特異度高,靈敏度與rRT-PCR相噹,有望應用于CV-A6感染的快速篩選,具有在基層醫療衛生機構和現場推廣與應用的潛力.
목적 건립기우간기내분람(hydroxy naphthol blue, HNB)안색변화적간단、령민화쾌속적배개도역전록등온확증기술(RT-LAMP)검측방법,응용우가살기병독A6형(coxsackievirus A6,CV-A6)적검측.방법 침대CV-A6적VP1기인설계6조특이인물,재등온조건하(63℃)진행50 min확증반응.확증전재반응체계중가입HNB,통과관찰안색변화진행검측결과판정.사용다충장도병독진행특이성험증,사용제도희석적체외전록CV-A6전VP1기인RNA진행령민도분석,동시여실시형광정량역전록PCR(rRT-PCR)검측결과진행비교,병대92빈수족구병환자림상표본진행검측.결과 본연구건립적RT-LAMP방법대제CV-A6외적23충장도병독적검측결과균위음성,령민도위100고패/반응,여rRT-PCR방법상당.재대92빈수족구병림상표본적검측중,검측결과여rRT-PCR방법상부,Kappa치위1,령민도화특이성균위100%.결론 본연구건립적침대CV-A6적LAMP검측방법,특이도고,령민도여rRT-PCR상당,유망응용우CV-A6감염적쾌속사선,구유재기층의료위생궤구화현장추엄여응용적잠력.
Objective To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).Methods The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 ℃ for 50 min.The products were detected through visual inspection of color change by the pre-addition of HNB dye.The specificity was validated by detecting a collection of different human enteroviruses.The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel.This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.Results A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change.The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR.The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR.The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.Conclusion The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection.It also has the potential to be used in resource-limited clinical sites and field study.