Wenjie Zheng

A loop-mediated isothermal amplification method targets the phoP gene for the detection of Salmonella in food samples

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Salmonella in food. The assay used specifically designed primers to target within the phoP gene and correctly identified all 66 strains of Salmonella and 73 non-Salmonella strains tested. The phoP gene regulates the expression of genes involved in virulence and the survival of Salmonella from destruction by macrophage. The probability of detection was 100% when a Salmonella cell suspension containing 10(1) CFU/ml was used as a template in the LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in buffered peptone water, followed by extraction and purification of DNA. In this way, 85 various food samples were investigated for Salmonella including minced meat of pig and raw milk. The diagnostic accuracy was shown to be 100% when compared to the traditional culture method. This combination of sample enrichment, and LAMP assay can detect 35 CFU per 250 ml of prepared food samples. The overall analysis time for the LAMP assay method was approximately 24 h. This is in contrast to 5 to 7 days of analysis time required for the traditional culture method. Consequently, the LAMP described here has the potential to become a standardized method for the rapid detection of Salmonella in diagnostic laboratories once further validated by inter-laboratory studies.

PCR detection of Klebsiella pneumoniae in infant formula based on 16S–23S internal transcribed spacer

A PCR detection based on 16S-23S rDNA internal transcribed spacer (ITS) of Klebsiella pneumoniae was developed in the present study. Nineteen different ITS sequences were amplified from 6 strains of K. pneumoniae by universal primers. By sequencing and alignment of these sequences to the other homologous in GenBank, species-specific primers of K. pneumoniae, Pf/Pr1 and Pf/Pr2, were designed for amplification of the ITS sequence from the operon containing tDNA(Ala) and tDNA(Ile). Ten type strains and 21 isolates of K. pneumoniae were positive to the PCR detection, and all of the non-K. pneumoniae reference strains (79 strains) were negative. The enrichment was performed in this procedure with a modified growth media to enrich K. pneumoniae from 1.5 CFU/100 g infant formula to about 10(5) CFU/ml in 900 ml of the media. Combination of the enrichment, with the PCR assay can detect 1.5 CFU/100 g infant formula of K. pneumoniae within 48 h. Furthermore, K. pneumoniae strains KPE050803 and KPE 050830 were identified by this method in 63 infant formula samples.