Rapid confirmation of five non-typhoidal Salmonella (NTS) serotypes using a low-cost portable-DIY OpenPCR© thermocycler

Aims@#OpenPCR is a low cost yet accurate thermocycler which can be self-built. The aim of the study is to highlight a low-cost alternative method for rapid confirmation of five predominant non-typhoidal Salmonella (NTS) serotypes using a multiplex PCR on a portable-DIY OpenPCR© thermocycler. @*Methodology and results@# Eight multiplex polymerase chain reaction (mPCR) samples containing genomic DNA of S. Agona selectively placed on the wells of the conventional PCR and OpenPCR© thermocyclers showed uniform heating in both thermocyclers. The limit of detection was similar for both thermocyclers for all five serotypes. The limit of detection for S. Typhimurium, S. Agona and S. Weltevreden was 10 pg/µL whereas the limit of detection for S. Enteriditis and S. Heidelberg was 1 pg/µL and 100 pg/µL, respectively. This assay incorporated a panel of unique genes; STM4495, SEN1392, SeHa-C4893, SeAg-B1096 and SENTW-3241 which were previously identified to be specific for S. Typhimurium, S. Enteritidis, S. Heidelberg, S. Agona, and S. Weltevreden, respectively, as well as the pan-Salmonella gene invA as internal control (IC) and pan-bacteria gene 16S rRNA to serve as amplification control (AC). The analytical specificity of the mPCR assay was found to be 100% for all five NTS using OpenPCR© thermocyclers. @*Conclusion, significance and impact of study@#The feasibility and low cost of the OpenPCR© thermocycler makes this device an ideal alternative for mPCR assay for rapid confirmation of NTS serotypes.

The Development of Rapid PCR / 中国生物工程杂志

Polymerase chain reaction (PCR) is one of the common techniques in molecular biology, which can amplify nucleic acids through the cycle of denaturation, annealing and extension. Based on the principle of common PCR, rapid PCR is to realize the amplification of nucleic acids in less time without affecting the specificity, sensitivity and fidelity of the reaction. A lot of research work in this field has been going on in recent years. This article will make a review of the development of rapid PCR with emphases on the improvement of DNA polymerase, the choice of additives and the improvement of thermocyclers.

Detection of Serum HCV RNA by Rapid Cycle PCR Using Hot Air Thermocycler with Capillary Tubes / 대한임상미생물학회지

BACKGROUND: Reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of Hepatitis C virus (HCV) RNA from serum. The PCR by conventional heat block thermocycler using small plastic tube is time and reagent consuming procedure, but rapid cycle PCR (RPCR) by hot air thermocycler using glass capillary tube is very rapid and economic. Therefore, RPCR have been recognized as a very convenient method for routine diagnostic test in clinical laboratories, but there are few reports about its usage for the detection of HCV RNA. METHODS: We selected two sets of primer pair from 5'noncoding region of HCV RNA genome, and optimized RPCR condition using hot air rapid thermocycler with master mix in capillary tubes. And RT-RPCR for detection of HCV RNA were performed on the serum of 58 patients, which were tested anti -HCV antibody by EIA. RESULTS: The optimized RPCR conditions were: denaturation; 94 degrees C for "0" sec, annealing; 55 degrees C (first) and 50 degrees C (nested) for "0" sec, elongation; 72 degrees C for "0" sec, and amplification cycles were 30 cycles. The consuming times per cycle were 30 sec (first) and 40 sec (nested), respecti vely, so the total involving times for nested RPCR were 35 min. Of the 42 EIA positive samples, 26 (62%) were RT-RPCR positive. CONCLUSIONS: RT-RPCR using hot air thermocycler with glass capillary tubes for detection of HCV RNA in serum is very rapid and economic than conventional PCR using heat block thermocycler. Therefore HCV RNA detection by RT-RPCR appears to be very useful for routine clinical laboratory diagnostic method.