{"title":"[Rapid detection of mutations related to Mycobacterium leprae drug resistance by using Hp-rPCR (hairpin primer- real time PCR) method].","authors":"Masanori Kai","doi":"10.5025/hansen.83.6","DOIUrl":null,"url":null,"abstract":"<p><p>Rapid and simple detection method of drug resistance bacteria is required. In the present study, Hp-rPCR (hairpin primer-real time PCR) was applied to Mycobacterium leprae genes to detect mutations. Target sites of the method were as follows: first base and second base on 53rd codon and second base on 55th codon infolP1 gene for dapsone resistance, first base on 441st codon and 451st codon and second base on 456th and 458th codon in rpoB gene for rifampicin resistance, and first base on 89th codon and second base on 91st codon in gyrA gene for quinolone resistance which were common mutation sites in clinical reports. The total number of the target sites was 9. Mycobacterium leprae, Thai-53, Zensho-2 and Zensho-4 were used as reference bacteria in the present study and clear, reliable results were obtained. Double-blind study was conducted using 15 samples. The number of target sites was calculated as 135 in total by 9 sites in 15 samples. There was only one misreading in the blind samples and the sensitivity was more than 99%.</p>","PeriodicalId":35918,"journal":{"name":"Japanese Journal of Leprosy","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2014-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.5025/hansen.83.6","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Japanese Journal of Leprosy","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.5025/hansen.83.6","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Medicine","Score":null,"Total":0}
引用次数: 0
Abstract
Rapid and simple detection method of drug resistance bacteria is required. In the present study, Hp-rPCR (hairpin primer-real time PCR) was applied to Mycobacterium leprae genes to detect mutations. Target sites of the method were as follows: first base and second base on 53rd codon and second base on 55th codon infolP1 gene for dapsone resistance, first base on 441st codon and 451st codon and second base on 456th and 458th codon in rpoB gene for rifampicin resistance, and first base on 89th codon and second base on 91st codon in gyrA gene for quinolone resistance which were common mutation sites in clinical reports. The total number of the target sites was 9. Mycobacterium leprae, Thai-53, Zensho-2 and Zensho-4 were used as reference bacteria in the present study and clear, reliable results were obtained. Double-blind study was conducted using 15 samples. The number of target sites was calculated as 135 in total by 9 sites in 15 samples. There was only one misreading in the blind samples and the sensitivity was more than 99%.
需要快速简便的耐药菌检测方法。本研究采用发夹引物实时荧光定量PCR (hairpin引物-real - time PCR)技术检测麻风分枝杆菌基因突变。方法的靶位点为:folp1基因的第53密码子第1碱基、第2碱基和第55密码子第2碱基为氨苯松耐药位点;rpoB基因的第441密码子第1碱基、第451密码子第1碱基、第456密码子第2碱基、第458密码子第1碱基为利福平耐药位点;gyrA基因的第89密码子第1碱基和第91密码子第2碱基为喹诺酮类耐药位点,这是临床报道中常见的突变位点。目标站点总数为9个。本研究以麻风分枝杆菌Thai-53、zensho2和zensho4为参比菌,得到了清晰、可靠的结果。采用双盲法对15例样本进行研究。15个样本中9个位点共计算出135个目标位点。盲检样本中仅有一次误读,灵敏度大于99%。