Application of a Duplex PCR Approach for the Specific and Simultaneous Detection of Clostridium Spp. and Lactobacillus Spp. in Broiler Gastrointestinal Tract

摘要

Detection of Clostridium spp. and Lactobacillus spp. in gastrointestinal contents of broiler by conventional culture-based microbiological methods is laborious and time-consuming. A novel method for the reliable detection of Clostridium spp. and Lactobacillus spp. was designed in the form of a rapid duplex PCR method that allowed the simultaneous detection, in a single tube, of two common bacterial genera in broiler gastrointestinal tract using four primers (Clos58-f, Clos780-r, LAA-f and LAA-r) targeting 16S rDNA sequence. Results showed that there were Clostridium spp. and Lactobacillus spp. in all four gastrointestinal segments viz. duodenum, jejunum, ileum and caecum of broilers and no cross detection of other strains occurred. This duplex PCR produced two distinct bands, of 722-bp and 286-bp, for Clostridium spp. and Lactobacillus spp. respectively. The 722-bp band produced by two (Clos58-f and Clos780-r) of four primers in duplex PCR reaction, was specific only from Clostridium spp. and not obtained from other non-target microorganisms. On the other hand, the 286-bp band was produced by two primers, LAA-f and LAA-r, and was specific for the Lactobacillus spp. genus. The divergence between the size of the Clostridium spp. (722 bp) and Lactobacillus spp. (286 bp) was suitable for developing the duplex PCR assay. As Clostridium spp. and Lactobacillus spp. can be dominant genera in the chicken gut, this method could be very useful to analyze microbial populations in chicken gut or possibly other similar samples.