Improvement and evaluation of loop-mediated isothermal amplification combined with a chromatographic flow dipstick assay and utilization in detection of Vibrio cholerae

Loop-mediated isothermal amplification (LAMP) is a specific, sensitive, and easy-to-perform nucleic acid analytical technique with wide application for diagnosis of disease. Recently, LAMP combined with use of a lateral chromatographic flow dipstick (LFD) has been widely used in nucleic acid detection. However, the LFD mechanism has not been systematically analyzed, and the optimal combination of labeled primers has not been adequately evaluated. We analyzed the LAMP mechanism and discovered that the labeled loop primers played a significant role in the LFD assay. To verify our hypothesis, we developed two LFD assays for Vibrio cholerae to detect the ctxA gene and the 16S–23S ribosomal DNA internal transcribed spacer (ITS). We labeled the inner primers [forward inner primer (FIP) and backward inner primer (BIP)] and loop primers [forward loop primer (LF) and backward loop primer (LB)]. Then the labeled and unlabeled primers were combined to form ten different primer sets. We assessed the specificity, sensitivity, and efficiency of LFD assays with use of different primer compositions. All triple-labeled primer sets resulted in false positive results in the LFD assay, as did the FIP and BIP double-labeled primer set. Other double-labeled-primer sets used in LFD assays showed higher sensitivity than the LAMP assays. Moreover, FIP and LF double-labeled and BIP and LB double-labeled sets had the highest sensitivity. In both cases, assays could be performed in 20 min. We also applied the ITS LFD assays in food samples. The enrichment broths of 112 oyster samples were tested, and the proportion that tested positive by the LFD assays was 6.25%, which was not lower than the rate for the conventional PCR method (5.36%). Graphical abstract ᅟ