Shortening distance of forward and reverse primers for nucleic acid isothermal amplification.

Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60-65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40-60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template.

Rapid detection of Vibrio cholerae by loop mediated isothermal amplification (LAMP) method / 生物工程学报

Vibrio cholerae is an important foodborne pathogen, mainly causes acute intestinal infectious disease. The development of rapid method for detecting Vibrio cholerae is critical for early diagnosis of its infection. In this study, two pairs of specific primers were designed according to housekeeping gene mdh of Vibrio cholerae. Following optimization of the reaction, DNA loop-mediated isothermal amplification (LAMP) for rapidly detecting Vibrio cholerae was successfully established. The optimal reaction for the LAMP assay is 65 degrees C for 60 min, with detection limit for cultivated Vibrio cholerae of 25 CFU/mL and for its contaminated food of 32 CFU/g. The specificity of the assay was determined using thirty-three kinds of same species or closely related bacteria, only Vibrio cholerae strains were specifically amplified. In practice, 85 pieces of positive samples were detected from 1057 pieces of shrimps, crabs, oysters, meat and human diarrhea complex using the LAMP method, which accorded with the detection result by ISO TS 21872-1-2007. Thus, the LAMP assay established in this study is a sensitive, rapid and simple tool for detecting Vibrio cholerae and will facilitate the surveillance for its control.