Development and In-House Validation of a Real-Time Polymerase Chain Reaction for the Detection of Listeria monocytogenes in Meat.

Listeriosis is a foodborne disease caused by Listeria monocytogenes. The aims of this work were to develop and validate an in-house real-time polymerase chain reaction (RT-PCR) for the detection of L. monocytogenes, and to determine its prevalence in raw ground beef samples from 53 butcheries that also sell ready-to-eat foods. One set of primers and one hydrolysis probe were designed for hly gene detection and then challenged with pure strains. The detection was successful for all L. monocytogenes strains analyzed and negative for all non-L. monocytogenes strains (detection limit, 10 colony forming unit [CFU]/mL). Inclusivity, exclusivity, and analytical accuracy were 100%. L. monocytogenes was detected in 41.5% of raw ground beef samples from the 53 butcheries analyzed. This RT-PCR may be a valuable method for rapid detection of L. monocytogenes in meat.

Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

Association of a region of bovine chromosome 1 (BTA1) with age at puberty in Angus bulls.

Age at puberty is an important component of reproductive performance in cattle, so it is important to identify genes that contribute to the regulation of the onset of puberty and polymorphisms that explain differences between bulls. In a previous study, we found putative associations between age at puberty in Angus bulls and single-nucleotide polymorphisms (SNPs) in Chromosomes 1 and X. In the present work we aimed to confirm these findings in a larger sample of Angus bulls (n = 276). Four SNPs located in these regions were genotyped using SEQUENOM technology and the genotypes obtained were tested for association with age at puberty. The results showed that SNPs rs135953349 and rs110604205 on BTA1 were still significantly associated with age of puberty estimated at progressive sperm motility of 10% (P < 0.05). The association previously found on Chromosome X could not be confirmed. Analysis of the bovine genome revealed that the associated region (99.17-99.99 Mb) contained four predicted loci: myelodysplasia syndrome 1 (MDS1) and ecotropic virus integration site 1 (EVI1) complex locus (MECOM), eGF-like and EMI domain-containing 1 pseudogene-like (LOC100337483), microRNA mir-551b (MIR551B) and mCG140927-like (LOC100139843). The results obtained could contribute to the understanding of puberty regulation and could be useful for further identification and annotation of gene function in the context of reproduction.

Effectiveness of single-nucleotide polymorphisms to investigate cattle rustling.

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single-nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32-SNP panel genotyped through OpenArrays(TM) (real-time PCR based) was evaluated to resolve cattle-specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32-SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.

Comparison of the effectiveness of microsatellites and SNP panels for genetic identification, traceability and assessment of parentage in an inbred Angus herd.

During the last decade, microsatellites (short tandem repeats or STRs) have been successfully used for animal genetic identification, traceability and paternity, although in recent year single nucleotide polymorphisms (SNPs) have been increasingly used for this purpose. An efficient SNP identification system requires a marker set with enough power to identify individuals and their parents. Genetic diagnostics generally include the analysis of related animals. In this work, the degree of information provided by SNPs for a consanguineous herd of cattle was compared with that provided by STRs. Thirty-six closely related Angus cattle were genotyped for 18 STRs and 116 SNPs. Cumulative SNPs exclusion power values (Q) for paternity and sample matching probability (MP) yielded values greater than 0.9998 and 4.32E(-42), respectively. Generally 2-3 SNPs per STR were needed to obtain an equivalent Q value. The MP showed that 24 SNPs were equivalent to the ISAG (International Society for Animal Genetics) minimal recommended set of 12 STRs (MP ∼ 10(-11)). These results provide valuable genetic data that support the consensus SNP panel for bovine genetic identification developed by the Parentage Recording Working Group of ICAR (International Committee for Animal Recording).

Association between GNRHR, LHR and IGF1 polymorphisms and timing of puberty in male Angus cattle.

BACKGROUND: In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Although the timing of puberty is likely to be a multigenic trait, previous studies indicate that there may also be single genes that exert major effects on the timing of puberty within the general population. Despite its economic importance, there are not many SNPs or genetic markers associated with the age of puberty in male cattle. In the present work, we selected three candidate genes, GNRHR, LHR and IGF1, and associated their polymorphisms with the age of puberty in Angus male cattle. RESULTS: After weaning, 276 Angus males were measured every month for weight (W), scrotal circumference (SC), sperm concentration (C) and percentage of motility (M). A total of 4 SNPs, two within GNRHR, one in LHR and one in IGF1 were genotyped using the pyrosequencing technique. IGF1-SnaBI SNP was significant associated (P < 0.01) with age at SC 28 cm, but it were not associated with age at M 10% and C 50 million. Genotype CC exhibited an average age at SC 28 cm of 7 and 11 days higher than CT (p = 0.037) and TT (p = 0.012), respectively. This SNP explained 1.5% of the genetic variance of age of puberty at SC28. LHR-I499L, GNRHR-SNP5 and GNRHR-SNP6 were not associated with any of the measurements. However, GNRHR haplotypes showed a suggestive association with age at SC 28 cm. CONCLUSIONS: The findings presented here could support the hypothesis that IGF1 is a regulator of the arrival to puberty in male calves and is involved in the events that precede and initiate puberty in bull calves. Given that most studies in cattle, as well as in other mammals, were done in female, the present results are the first evidence of markers associated with age at puberty in male cattle.

Implication of population structure in the resolution of cattle stealing cases.

Estimation of population subdivision using genetic markers shows that genetic differentiation in livestock and pet breeds is significantly higher than in human populations. Nevertheless, the influence of population substructure and sample size on match probability has not been extensively analyzed in domestic species. To evaluate the magnitude of the subpopulation effect on estimation of match probabilities in bovine robbery cases, we calculated and compared the match probabilities obtained from cattle breed databases using both real, adjudicated cases from the Buenos Aires Province (Argentina), as well as simulated data. While the Balding and Nichols' correction, when applied to the population database used in the case, produce a more conservative value favorable to the defendant, the match probabilities calculated using the simple product estimator produce a value favorable to the prosecution. We suggest an alternative procedure that can be used. The method consists of choosing the highest value from all match probabilities calculated from the database of each breed. This approach represents an intermediate and more accurate estimation of match probability, although it still produces a slight conservative value favorable to the defense.

A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene.

We developed a simple, quick assay in order to discriminate forensic samples among human, and common domestic and livestock species of the Pampean region, Argentina. A mitochondrial cytochrome b fragment amplified with universal primers was separately digested with three restriction enzymes (AluI, HaeIII, and HinfI) and the resulting fragments were resolved through electrophoresis in polyacrylamide gels. This PCR-RFLP method allowed us to identify the target species and worked on degraded samples. The assay was successfully applied in livestock robbery cases in Argentine, and may be useful when attempting a first assessment as to the specific status of a forensic evidence.