Comparison of Bacterial Culture and Real‐Time PCR for the Detection ofSalmonellain Grow–Finish Pigs in Western Canada Using a Bayesian Approach

Abstract

Impacts• Using a Bayesian model specifying dependence between tests, RT-PCR sensitivity (Se) and specificity (Sp) were estimated to be 90% (95% probability interval 74, 97) and 99% (98, 99), respectively, while culture Se was 92% (75, 99).• The correlation between RT-PCR and bacterial culture was found to bias estimates of test accuracy; researchers should adjust for this correlation during analysis.• The RT-PCR evaluated in this study performed comparably to culture and may be a useful alternative to culture for screening large numbers of samples, particularly when Salmonella prevalence is low. SummaryThe study objective was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) and a culture protocol used to detect Salmonella in the faeces of grow-finish pigs using a Bayesian approach. The RT-PCR was invA-gene-based assay, while the culture protocol included pre-enrichment in buffered peptone water, selective enrichment in tetrathionate and RappaportVassiliadis broths, and isolation on semi-solid (modified semi-solid RV) or solid (XLT4, Rambach) agar plates. Bayesian analysis was performed using a two-test, two-population model with dependence between culture and RT-PCR and compared to a second model with conditional independence between these two tests. Two hundred and ninety three individual faecal and 294 pooled pen samples from grow-finish pig collected from 10 farms were tested and results were divided into two groups according to herd size (five herds <250 sows, five herds with >400 sows). In the dependence model, RT-PCR sensitivity (Se) and specificity (Sp) were estimated to be 90% (95% probability interval 74, 97) and 99% (98, 99), respectively. Culture Se was 92% (75, 99), while culture Sp was considered 100% as all culture-positive samples were confirmed by serotyping.In the conditional independence model, RT-PCR Se and Sp, and culture Se, were 96% (93, 98), 99% (98, 100) and 97% (94, 100), respectively. The dependence model resulted in posterior estimates of Se that were lower and with broader probability intervals than the independence model, indicating that when RT-PCR and culture are evaluated relative to each other, the correlation between these tests is an important source of bias and should be adjusted for during analysis. The RT-PCR evaluated in this study performed almost comparably to culture; given the cost savings associated with using this test and more timely results, the RT-PCR may be a useful alternative to culture for screening large numbers of samples, particularly when Salmonella prevalence is low.

Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food

“…If previously designed primer pairs listed in Table 3 are used, several PCR assays would fail to detect numerous Salmonella spp., whose sequences are currently available in GenBank. This could partially explain the false-negative results encountered in Salmonella detection [36]. At the same time, although invA is capable of excluding non- Salmonella strains, our BLAST sequence analysis of invA demonstrated that some non- Salmonella groups such as E. coli, Staphylococcus aureus subsp.…”

“…In general, the invA- based PCR assays provide higher sensitivity, shorter turnaround time, and reduced labor cost, making it an excellent alternative to conventional culture method for pathogen detection [29]. However, conspicuous variations in sensitivity and specificity of invA- based PCR assays have been documented by numerous studies [1,29-35], and one of the possible reasons for such discordant outcomes may be due to the use of different primers for gene detection in the assays such as conventional or qPCR [36]. In an effort to better understand the variations caused by the usage of different primers for gene detection in PCR assays, we systematically evaluated the most commonly used invA primer pairs for the detection of Salmonella in thirteen (n = 13) PCR assays (Table 3; Figure 4).…”

Sensitivity of the ISO 6579:2002/Amd 1:2007 Standard Method for Detection of Salmonella spp. on Mesenteric Lymph Nodes from Slaughter Pigs

“…Thus, it was initially assumed that the Se ISO and the Se PCR were conditionally independent; i.e., the probability of a positive result for culture is the same regardless of the result obtained for the PCR and vice versa. However, since both tests were performed on the same BPW-enriched samples, some degree of Se correlation between tests was not unexpected (40). Hence, a conditional independence model for two tests was initially carried out in two populations (29) using Winbugs software (http://www.mrc-bsu.cam.ac.uk/bugs/).…”

Pathogenicity of Salmonella Strains Isolated from Egg Shells and the Layer Farm Environment in Australia

“…It is essential to note that qPCR methods detect both viable and nonviable bacteria while culture methods detect only viable bacteria. Such disparity in culture versus qPCR results has also been observed during Salmonella detection/isolation from swine feces (33).…”