Designing primers and evaluation of the efficiency of propidium monoazide – Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius

AbstractThe purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4–5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable

The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains

The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella

Designing a biochip following multiplex polymerase chain reaction for the detection of Salmonella serovars Typhimurium, Enteritidis, Infantis, Hadar, and Virchow in poultry products

Salmonella-contaminated foods, especially poultry-derived foods (eggs, chicken meat), are the major source of salmonellosis. Not only in the European Union (EU), but also in the United States, Japan, and other countries, has salmonellosis been an issue of concern for food safety control agencies. In 2005, EU regulation 1003/2005 set a target for the control and reduction of five target Salmonella enterica serovars—S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar, and S. Virchow—in breeding flocks. Thus, a simple biochip for the rapid detection of any of these five Salmonella serovars in poultry products may be required. The objectives of this study were to design S. Virchow-specific primers and to develop a biochip for the simultaneous identification of all or any of these five Salmonella serovars in poultry and poultry products. Experimentally, we designed novel polymerase chain reaction (PCR) primers for the specific detection of S. Virchow, S. Infantis, and S. Hadar. The specificity of all these primers and two known primer sets for S. Typhimurium and S. Enteritidis was then confirmed under the same PCR conditions using 57 target strains and 112 nontarget Salmonella strains as well as 103 non-Salmonella strains. Following multiplex PCR, strains of any of these five Salmonella serovars could be detected by a chromogenic biochip deployed with DNA probes specific to these five Salmonella serovars. In comparison with the multiplex PCR methods, the biochip assay could improve the detection limit of each of the Salmonella serovars from N×103 cfu/mL to N×102 cfu/mL sample in either the pure culture or the chicken meat samples. With an 8-hour enrichment step, the detection limit could reach up to N×100 cfu/mL

Development and application of tuf gene-based PCR and PCR-DGGE methods for the detection of 16 Bifidobacterium species

AbstractA total of 16 Bifidobacterium species were assayed by polymerase chain reaction (PCR) and PCR–denaturing gradient gel electrophoresis (PCR–DGGE) methods targeted on a 770-bp region of the tuf gene. Based on this sequence, a genus-specific primer set and 12 primer sets for 12 Bifidobacterium species including those previously reported for six probiotic species were developed. On the other hand, when these 16 Bifidobacterium species were subjected to PCR–DGGE analysis, 13 product migration patterns were obtained. PCR products for strains in pairs of B. adolescentis/B. thermophilum, B. longum/B. magnum and B. lactis/B. gallinarum migrated the same distance on the DGGE gel. Combined with species-specific PCR primers specific to B. adolescentis, B. longum and B. lactis, all of the 16 Bifidobacterium species could be identified. In addition, the subspecies of B. animalis, i.e., B. animalis and B. lactis, could be discriminated. This study indicated that the tuf gene is highly useful for the molecular detection of different Bifidobacterium species. Using the PCR and PCR–DGGE methods, 16 Bifidobacterium species, including those from probiotic products and those from other origins, could be rapidly identified