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Updated: May 8, 2026
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Published on: August 6, 2011
Multiplex PCR targeting slpA: a rapid screening method to predict common Clostridium difficile ribotypes among
Kelin Xiao1, Fanrong Kong, Qinning Wang
1*Shenzhen Bao'an Maternal and Child Health Hospital, Shenzhen, China †Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital ‡Sydney Emerging Infections and Biosecurity Institute, University of Sydney, Sydney, New South Wales, Australia §these authors contributed equally to the work.
Aims:
Based on the relationship between Clostridium difficile surface layer protein A (slpA) sequence types (STs) and PCR-ribotypes (RTs), a multiplex polymerase chain reaction (mPCR) assay was developed to rapidly confirm C. difficile toxigenicity and, simultaneously, to identify any of five slpA STs, gr, hr, fr, gc8 and 078, that usually correspond with globally distributed RTs, 001, 014, 017, 027 and 078, respectively.
Methods:
The mPCR, containing five slpA type-specific primers, was developed using 46 well-characterised C. difficile reference strains, representing 11 slpA STs, and validated by testing 90C. difficile clinical isolates.
Results:
The slpA mPCR correctly identified the five slpA STs without cross-reactions. A much higher proportion of moxifloxacin resistant (32/39; 82%) than susceptible (12/51; 24%) clinical isolates were slpA typeable (χ=30.3, p<0.0001), even when RT027 isolates were excluded [10/17 (59%) versus 12/51 (24%); χ=7.3, p=0.0071<0.01]. slpA mPCR correctly predicted the RTs of all 39 isolates that belonged to the five targeted RTs.
Conclusion:
slpA mPCR is simple, rapid and inexpensive. It can provisionally identify five globally significant, highly transmissible RTs, particularly among moxifloxacin resistant C. difficile isolates, and could be easily modified to include a broader range of slpA sequence types, based on local requirements.
