中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2008年
1期
48-52
,共5页
分枝杆菌,结核%微生物敏感性试验%基因突变%高效液相色谱法
分枝桿菌,結覈%微生物敏感性試驗%基因突變%高效液相色譜法
분지간균,결핵%미생물민감성시험%기인돌변%고효액상색보법
Mycobacterium tuberculosis%Mlcrobial sensitivity test%Gene mutation%High-performance liquid chromatography
目的 分析我国结核分枝杆菌氟喹诺酮类耐药基因gyrA突变的特点并探讨变性高效液相色谱法(DHPLC)的应用价值.方法 以结核分枝杆菌临床分离株109株(常规药敏试验显示87株氧氟沙星耐药,22株敏感)为对象,测定氧氟沙星最小抑菌浓度(MIC),同时进行gyrA基因氟喹诺酮类耐药决定区域(QRDR)DNA测序及DHPLC分析.结果 发现我国耐氧氟沙星结核分枝杆菌近来出现两个新的特点:一是gyrA QRDR双位点突变率高(87株耐药株中56.3%携有双位点突变);二是出现了以往认为的仅存在于其他细菌而在结核分枝杆菌中从未报道过的Ala74Ser突变(49株双位点突变耐药株中有20.4%携有该突变).gyrA基因突变类型与MIC之间并无密切关系.DHPLC测定中除常规的M.tb H37Rv以外还引入了M.tb Erdman做为参照菌株,成功地摆脱了95位AGC:ACC自然多态性的影响.与测序法相比较,DHPLC法的敏感度与特异度均达100%.结论 我国结核分枝杆菌氟喹诺酮类耐药已很严重,gyrA基因QRDR双位点突变率高为其特点.本研究建立的结核分枝杆菌gyrA基因突变的DHPLC分析法简便快速,稳定灵敏,在大面积大样本耐药监测以及临床快速药敏检测中有应用价值.
目的 分析我國結覈分枝桿菌氟喹諾酮類耐藥基因gyrA突變的特點併探討變性高效液相色譜法(DHPLC)的應用價值.方法 以結覈分枝桿菌臨床分離株109株(常規藥敏試驗顯示87株氧氟沙星耐藥,22株敏感)為對象,測定氧氟沙星最小抑菌濃度(MIC),同時進行gyrA基因氟喹諾酮類耐藥決定區域(QRDR)DNA測序及DHPLC分析.結果 髮現我國耐氧氟沙星結覈分枝桿菌近來齣現兩箇新的特點:一是gyrA QRDR雙位點突變率高(87株耐藥株中56.3%攜有雙位點突變);二是齣現瞭以往認為的僅存在于其他細菌而在結覈分枝桿菌中從未報道過的Ala74Ser突變(49株雙位點突變耐藥株中有20.4%攜有該突變).gyrA基因突變類型與MIC之間併無密切關繫.DHPLC測定中除常規的M.tb H37Rv以外還引入瞭M.tb Erdman做為參照菌株,成功地襬脫瞭95位AGC:ACC自然多態性的影響.與測序法相比較,DHPLC法的敏感度與特異度均達100%.結論 我國結覈分枝桿菌氟喹諾酮類耐藥已很嚴重,gyrA基因QRDR雙位點突變率高為其特點.本研究建立的結覈分枝桿菌gyrA基因突變的DHPLC分析法簡便快速,穩定靈敏,在大麵積大樣本耐藥鑑測以及臨床快速藥敏檢測中有應用價值.
목적 분석아국결핵분지간균불규낙동류내약기인gyrA돌변적특점병탐토변성고효액상색보법(DHPLC)적응용개치.방법 이결핵분지간균림상분리주109주(상규약민시험현시87주양불사성내약,22주민감)위대상,측정양불사성최소억균농도(MIC),동시진행gyrA기인불규낙동류내약결정구역(QRDR)DNA측서급DHPLC분석.결과 발현아국내양불사성결핵분지간균근래출현량개신적특점:일시gyrA QRDR쌍위점돌변솔고(87주내약주중56.3%휴유쌍위점돌변);이시출현료이왕인위적부존재우기타세균이재결핵분지간균중종미보도과적Ala74Ser돌변(49주쌍위점돌변내약주중유20.4%휴유해돌변).gyrA기인돌변류형여MIC지간병무밀절관계.DHPLC측정중제상규적M.tb H37Rv이외환인입료M.tb Erdman주위삼조균주,성공지파탈료95위AGC:ACC자연다태성적영향.여측서법상비교,DHPLC법적민감도여특이도균체100%.결론 아국결핵분지간균불규낙동류내약이흔엄중,gyrA기인QRDR쌍위점돌변솔고위기특점.본연구건립적결핵분지간균gyrA기인돌변적DHPLC분석법간편쾌속,은정령민,재대면적대양본내약감측이급림상쾌속약민검측중유응용개치.
Objective To analyze the characteristic of fluoroquinolone resistance gene gyrA mutation in Mycobacterium tuberculosis and to evaluate the value of denaturing HPLC(DHPLC) method.Methods One hundred and nine Mycobacterium tuberculosis clinical isolates among which 87 were found ofloxacin-resistant while 22 susceptible according to routine method,received minimal inhibitory concentration (MIC) test,gyrA quinolone resistance determining region (QRDR) DNA sequencing and DHPLC analysis concurrently. Results Two new characteristics of ofloxacin-resistant Mycobacterium tuberculosis were found: one was the high rate of double mutations in gyrA QRDR (56.3% of 87 ofloxacin-resistant isolates were found carrying double mutations),the other was that among these double-mutated isolates,20.4% (10/49) harbored the Ala74Ser mutation,which previously was thought to exist only in other bacteria,and had never been found in Mycobacterium tuberculosis. No close relationship was observed between gyrA mutation type and ofloxacin MIC. By introducing M.tb H37Rv and M.tb Erdman two reference strains,interference from codon 95 AGC:ACC polymorphism was successfully avoided. Compared to DNA sequencing,the sensitivity and specificity of DHPLC method were all 100%.Conclusions Fluoroquinolone-resistance of Mycobacterium tuberculosis in our country is quite severe. The characteristic is the high rate of gyrA QRDR double point mutations. DHPLC method set up in this work is simple,rapid,stable,sensitive and may be valuable in large samples of drug-resistance survillence in large area.
