Development and verification of a duplex loop-mediated isothermal amplification assay for simultaneous detection of toxin A and toxin B of Clostridium difficile
10.13200/j.cnki.cjb.004403
- VernacularTitle:艰难梭菌毒素A和B双重环介导等温扩增检测方法的建立及验证
- Author:
XI Xiaoyan
- Publication Type:Journal Article
- Keywords:
Clostridium difficiletoxin A(TcdA);
Clostridium difficile toxin B(TcdB);
Duplex loop-mediated isothermal amplification(LAMP);
Toxin typing
- From:
Chinese Journal of Biologicals
2025;38(1):67-72+79
- CountryChina
- Language:Chinese
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Abstract:
Objective To develop a duplex loop-mediated isothermal amplification(LAMP) assay for rapid detection of Clostridium difficile toxin A(TcdA) and B(TcdB), so as to provide technical support for the rapid detection and toxin typing of Clostridium difficile(CD) infection.Methods According to the conserved sequences of TcdA and TcdB genes, two sets of LAMP primers were designed respectively, and duplex LAMP amplification was performed in the same system. By optimizing the reaction system and reaction conditions, a rapid LAMP detection method for TcdA and TcdB was developed. The specificity and sensitivity of this method were verified, and the melting curves of the LAMP amplified products were analyzed. The 18 stool samples from patients with diarrhea were detected by using the developed method.Results The duplex LAMP reaction conditions for good amplification of both TcdA and TcdB were as follows: Mg~(2+) concentration 6. 8 mmol/L, dNTPs concentration 1. 2 mmol/L, and amplification temperature 64 ℃. The amplification products were preliminarily detected by using SYBR Green I staining, and the target gene(bacteria) showed a bright green fluorescence, while the six other common intestinal pathogens(Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis,and Clostridium botulinum) exhibited no fluorescence. The sensitivity of the duplex LAMP system for detection of TcdA and TcdB was 10~0 and 10~1 plasmids/reaction respectively, and the exact target toxins contained in CD strains were determined by analyzing the characteristic peaks of the melting curves. Eight of 18 stool samples from patients with diarrhea were positive in preliminary detection, and the melting curve analysis showed that all of them contained TcdA and TcdB.Conclusion A duplex LAMP detection method for TcdA and TcdB has been developed with good sensitivity and specificity, which can be used for the rapid detection of CD infection and can realize the simultaneous rapid detection of TcdA and TcdB.
