Rapid detection of ermB gene in Clostridium difficile by loop-mediated isothermal amplification.
Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. Here, we describe a sensitive and rapid molecular method to detect ermB in C. difficile to contribute to the wider epidemiological study. Five sets of loop-mediated isothermal amplification (LAMP) primers were designed and optimized for rapid detection of ermB. The specificity and sensitivity of the primers for ermB were detected, and the ermB LAMP assay was compared to conventional PCR with 80 clinical isolates of C. difficile. Real-time monitoring of turbidity and chromogenic reaction were used to determine negative and positive results. A total of 26 pathogenic bacterial strains of different species were found to be negative for ermB, which indicated the high specificity of the primers. ermB was detected in 78.8 % (63/80) of the clinical isolates by both LAMP and conventional PCR. The detection limit of LAMP was 36.1  pg DNA μl- 1 and its sensitivity was 10-fold greater than that of conventional PCR. This study is the first report regarding the development and application of the LAMP assay for detection of the ermB gene in C. difficile strains. The developed LAMP method is sensitive, specific and provides a user-friendly visual approach for the rapid detection of ermB-bearing C. difficile.
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.|Infectious Disease Center, The Fifth Affiliated Hospital, Sun Yat-sen University, 52 Meihua East Road, Zhuhai 519000, PR China.
Minyi
M
Lin
Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 20 Dongda Street, Fengtai District, Beijing 100071, PR China.
Wei
W
Liu
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.
Pu
P
Wang
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.
Jiasheng
J
Tan
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.
Youlian
Y
Zhou
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.
Peiqun
P
Wu
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.
Ting
T
Zhang
Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 20 Dongda Street, Fengtai District, Beijing 100071, PR China.
Jing
J
Yuan
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangdong Provincial Key Laboratory of Gastroenterology, 1838 North Guangzhou Ave, Guangzhou 510515, PR China.
Ye
Y
Chen
D016360:Clostridioides difficile / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013997:Time Factors
D017931:DNA Primers; D004269:DNA, Bacterial; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase