中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2013年
6期
543-547
,共5页
王欢%黄庆%黄君富%夏涵%杨昭%府伟灵
王歡%黃慶%黃君富%夏涵%楊昭%府偉靈
왕환%황경%황군부%하함%양소%부위령
假单胞菌,铜绿%抗药性,细菌
假單胞菌,銅綠%抗藥性,細菌
가단포균,동록%항약성,세균
Pseudomonas aeruginosa%Drug resistance,bacterial
目的 建立肉眼直接判读检测结果的铜绿假单胞菌0prD2耐药基因环介导等温扩增(LAMP)快速检测方法.方法 对2011年12月至2012年6月西南医院微生物室收集的47株铜绿假单胞菌标本进行前瞻性研究.利用LAMP针对铜绿假单胞菌OprD2耐药基因设计两对引物,特异性识别OprD2的6个独立区域来快速扩增.通过在LAMP反应体系中加入核酸染料羟基萘酚蓝(HNB),建立可直接用肉眼判读检测结果的LAMP反应体系,并用该方法检测和分析47株铜绿假单胞菌OprD2耐药基因的分布情况及其与抗生素耐受性的相关性.结果 设计出针对铜绿假单胞菌OprD2耐药基因可直接肉眼判读结果的恒温扩增检测方法,其检测体系的灵敏度为17.414 μg/L,比常规PCR方法(灵敏度:174.14 μg/L)高10倍.49％ (23/47)的菌株OprD2基因缺失,OprD2基因缺失株的头孢噻肟、左氧氟沙星、氨曲南、哌拉西林、亚胺培南和美罗培南耐药率分别为100％(23/23)、57％ (13/23)、48％ (11/23)、48％(11/23)、48％ (11/23)和43％(10/23).与OprD2基因阳性株相比,OprD2基因缺失株的亚胺培南、左氧氟沙星和美罗培南耐药比例明显升高,差异有统计学意义(x2值分别为9.155、4.846、4.037,P值分别为0.002、0.028、0.045).结论 建立了可直接肉眼判读检测结果的铜绿假单胞菌OprD2耐药基因LAMP快速技术.OprD2基因缺失导致铜绿假单胞菌以亚胺培南为主的耐药,因此明确OprD2基因分布状况有助于临床抗菌药物的选用.
目的 建立肉眼直接判讀檢測結果的銅綠假單胞菌0prD2耐藥基因環介導等溫擴增(LAMP)快速檢測方法.方法 對2011年12月至2012年6月西南醫院微生物室收集的47株銅綠假單胞菌標本進行前瞻性研究.利用LAMP針對銅綠假單胞菌OprD2耐藥基因設計兩對引物,特異性識彆OprD2的6箇獨立區域來快速擴增.通過在LAMP反應體繫中加入覈痠染料羥基萘酚藍(HNB),建立可直接用肉眼判讀檢測結果的LAMP反應體繫,併用該方法檢測和分析47株銅綠假單胞菌OprD2耐藥基因的分佈情況及其與抗生素耐受性的相關性.結果 設計齣針對銅綠假單胞菌OprD2耐藥基因可直接肉眼判讀結果的恆溫擴增檢測方法,其檢測體繫的靈敏度為17.414 μg/L,比常規PCR方法(靈敏度:174.14 μg/L)高10倍.49％ (23/47)的菌株OprD2基因缺失,OprD2基因缺失株的頭孢噻肟、左氧氟沙星、氨麯南、哌拉西林、亞胺培南和美囉培南耐藥率分彆為100％(23/23)、57％ (13/23)、48％ (11/23)、48％(11/23)、48％ (11/23)和43％(10/23).與OprD2基因暘性株相比,OprD2基因缺失株的亞胺培南、左氧氟沙星和美囉培南耐藥比例明顯升高,差異有統計學意義(x2值分彆為9.155、4.846、4.037,P值分彆為0.002、0.028、0.045).結論 建立瞭可直接肉眼判讀檢測結果的銅綠假單胞菌OprD2耐藥基因LAMP快速技術.OprD2基因缺失導緻銅綠假單胞菌以亞胺培南為主的耐藥,因此明確OprD2基因分佈狀況有助于臨床抗菌藥物的選用.
목적 건립육안직접판독검측결과적동록가단포균0prD2내약기인배개도등온확증(LAMP)쾌속검측방법.방법 대2011년12월지2012년6월서남의원미생물실수집적47주동록가단포균표본진행전첨성연구.이용LAMP침대동록가단포균OprD2내약기인설계량대인물,특이성식별OprD2적6개독립구역래쾌속확증.통과재LAMP반응체계중가입핵산염료간기내분람(HNB),건립가직접용육안판독검측결과적LAMP반응체계,병용해방법검측화분석47주동록가단포균OprD2내약기인적분포정황급기여항생소내수성적상관성.결과 설계출침대동록가단포균OprD2내약기인가직접육안판독결과적항온확증검측방법,기검측체계적령민도위17.414 μg/L,비상규PCR방법(령민도:174.14 μg/L)고10배.49％ (23/47)적균주OprD2기인결실,OprD2기인결실주적두포새우、좌양불사성、안곡남、고랍서림、아알배남화미라배남내약솔분별위100％(23/23)、57％ (13/23)、48％ (11/23)、48％(11/23)、48％ (11/23)화43％(10/23).여OprD2기인양성주상비,OprD2기인결실주적아알배남、좌양불사성화미라배남내약비례명현승고,차이유통계학의의(x2치분별위9.155、4.846、4.037,P치분별위0.002、0.028、0.045).결론 건립료가직접육안판독검측결과적동록가단포균OprD2내약기인LAMP쾌속기술.OprD2기인결실도치동록가단포균이아알배남위주적내약,인차명학OprD2기인분포상황유조우림상항균약물적선용.
Objective To establish a rapid detection approach by visual interpretation directly for OprD2 resistance gene of Pseudomonas aeruginosa based on the Loop-mediated isothermal amplification (LAMP),and provide a quick and effective method for clinical monitoring of Pseudomonas aeruginosa strains.Methods Totally 47 strains of Pseudomonas aeruginosa collected from December 2011 to June 2012 in Southwest Hospital of microorganisms were prospectively studied.Four LAMP primers (two inner,two outer) were designed according to the six zones of the OprD2 gene of Pseudomonas aeruginosa.A positive reaction is indicated by the color change after adding an intercalating dye (hydroxy naphthol blue) to the reaction solution.This method was used to detect and analyze the distribution of OprD2 resistance gene in 47 strains of Pseudomonas aeruginosa and its coirelation with antibiotic resistance.Results The LAMP assays showed 100％ specificity for the OprD2 gene,and the sensitivity (with the lowest detection limits of 17.414 μg/L) was 10-fold higher than that of conventional PCR assays.The OprD2 gene was negative in 23 strains by both conventional PCR and LAMP.In OprD2 negative strains,the resistance rate of cefotaxime,levofloxacin,aztreonam,piperacillin,imipenem and meropenem was 100％ (23/23),57％ (13/23),48％ (11/23),48％ (11/23),48％ (11/23) and 43％ (10/23).Compared with the OprD2 positive strains,statistical analysis showed that the resistance rate of imipenem,levofloxacin and meropenem in OprD2 negative strains increased significantly (chisquare value is 9.155,4.846,4.037,P value was 0.002,0.028,0.045,and so there was significant difference).Conclusions The established LAMP approach in this study enables rapid,sensitive and specific detection of OprD2 gene in Pseudomonas aeruginosa by visual interpretation.Deficiency of OprD2 gene confers Pseudomonas aeruginosa a basal level of resistance to carbapenems especially to imipenem.The identification of OprD2 gene distribution in Pseudomonas aeruginosa is helpful to the selection of antimicrobial agents in the infection treatment.
