Rapid screening of Salmonella by loop-mediated isothermal amplification combined with lateral flow dipstick / 上海预防医学

Objective:To establish a specific and sensitive method using loop-mediated isothermal amplification for rapid screening of Salmonella. Methods:The invA gene sequence of Salmonella was downloaded from GenBank. After homology comparison with DNAMAN software， amplification primers were designed in the conserved region， and a LAMP-LFD detection method was established. The reaction system was optimized， and the specificity and sensitivity of the method were verified. Results:The sensitivity of this method to detect Salmonella DNA was up to 1.0×101 copies/μL. The positive rate of anal swabs was the same as that of fluorescent PCR. Meanwhile， LAMP-LFD was easy to operate and did not need expensive instruments. The detection result could be obtained within 30 minutes. Conclusion:The LAMP-LFD method established in this study is rapid， simple， sensitive and specific， which is suitable for rapid screening of Salmonella.

Estandarización de una PCR para la detección del gen invA de Salmonella spp. en lechuga / Standardization of a PRC method for the detection of the Salmonella spp. invA gene in lettuce

Salmonella spp. es un patógeno bacteriano muy importante causante de diarreas, que es transmitido tanto por la vía fecal-oral, como por alimentos y agua contaminados. En este trabajo se estandarizó una técnica de PCR en lechuga para la detección del gen invA de Salmonella spp.; dicho gen se relaciona con el proceso de invasión al epitelio intestinal. Con la PCR desarrollada en este trabajo se logró estandarizar un método que permite la amplificación del gen invA con una detección de 10² UFC/25 g. Este método acorta los tiempos de respuesta de los resultados presuntivos y brinda información complementaria al cultivo tradicional del patógeno. El estudio del gen invA establece el potencial patógeno del microorganismo presente en la muestra, lo que puede ser de utilidad para la salud pública.

Salmonella spp. is a very important bacterial pathogen that causes diarrhea and which is transmitted both through the fecal-oral pathway, as by contaminated food and water. In this study we standardized a PCR method in lettuce for the detection of the Salmonella spp. invA gene. This gene is related to the invasion of the intestinal epithelium process. With the PCR method developed in this study we were able to standardize a method which permits the amplification of the invA gene with a 10² CFU/25 g detection. This method shortens the response times of the presumptive results and gives complementary information to the traditional culture of the pathogen. The study of the invA gene establishes the pathogenic potential of the microorganism present in the sample, which can be useful for public health purposes.

Transcription and expression characteristics of Leptospira virulence-associated protein InvA / 中华微生物学和免疫学杂志

objective To determine the existence of virulence-associated invA gene in different genospeeies of Leptospira interrogans reference strains in China.and to understand the alterations of invA gene transcription and expression of L.interrogans strain Lai before or after infecting cells.Methods PCR was applied to detect the invA gene of four L.interrogans strains belonging to four different genospecies and L.biflexa strain Patoc Ⅰ.The entire invA genes from the L.interrogans strains were cloned and then sequenced.The prokaryotic expression system of invA gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed.Using Ni-NTA affinity chromatography,the target recombinant protein rInvA was extracted and purified.Rabbits were immunized with rInvA to obtain antiserum and the titer of antiserum was determined by immunodiffusion test.A model of L interrogans strain Lai infecting human embryo kidney cell line HEK293 was established to detect the alterations of invA gene transcription and expression of the leptospiral strain before or after infecting the host cells by real-time fluorescent quantitative RTPCR and western blot assay.Results All the four tested L.interrogans strains had invA gene whereas L.biflexa strain Patoc Ⅰ not.The similarity of nucleotide and putative amino acid sequences of invA genes from the four L.interrogans strains belonging four different genospecies were 99.33%-100%and 98.66%-100%,respectively.The constructed prokaryotic expression system could efficiently express rInvA and the immunodiffusion titer of rabbit anti-rInvA serum was 1:16.After L.interrogans strain Lai infecting HEK293 cells for 30 min or above,the microbe could adhere the surface of the cells.On the 30 min after the infection,the mRNA level of invA gene of L.interrogans strain Lai was remarkably upregulated,and on the 45 min after infection the mRNA level presented a peak value and then graduated decreased.On the 45 min or 60 min after L.interrogans strain Lai infecting HEK293 cells,InvA protein could be detectable but before infection or after infection for over 90 min the InvA protein expression was negative.Conclusion The invA gene is a unique gene for pathogenic L.interrogans.The invA gene of L interrogans has characteristics of cell-touched expression and transient expression,which may have a close correlation with adhering and invading host cells.