Use of Pooled Samples for the Detection of Salmonella in Feces by Polymerase Chain Reaction

Abstract

Abstract. Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa $ 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.

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

“…Overall, in both populations, the most prevalent serogroup was B, followed by C1 (Table 4). Although there did not appear to be large differences regarding the Salmonella serogroups between P A and P B , a higher variability was observed regarding serotypes, which might somewhat influence Se ISO , since different serotypes may exhibit different growth characteristics in the same enrichment and selective media (9,11,38). However, no differences with respect to Se PCR were expected, since the invA gene is considered present in all Salmonella spp.…”

Development of a multiplex PCR protocol for the detection of several bacteria causing acute diarrhea in pig

“…là một trong những tác nhân gây tiêu chảy phổ biến ở lợn trưởng thành, trong đó, hai chủng Salmonella chủ yếu gây bệnh là S. choleraesuis và S. typhimurium, chiếm hơn 65% tình trạng nhiễm Salmonella ở lợn. Có khoảng 200 gene độc tồn tại ở Salmonella, trong đó gene invA có vai trò quan trọng trong quá trình xâm nhiễm của Salmonella vào thể chủ [8,12].…”

“…là một trong những tác nhân gây tiêu chảy phổ biến ở lợn trưởng thành, trong đó, hai chủng Salmonella chủ yếu gây bệnh là S. choleraesuis và S. typhimurium, chiếm hơn 65% tình trạng nhiễm Salmonella ở lợn. Có khoảng 200 gene độc tồn tại ở Salmonella, trong đó gene invA có vai trò quan trọng trong quá trình xâm nhiễm của Salmonella vào thể chủ [8,12].Một số công trình nghiên cứu ở Việt Nam và trên thế giới nhằm vào những vi khuẩn này với nhiều mục đích khác nhau. Nguyễn Tuyên Quang (2007) [14] nghiên cứu xác định các yếu tố gây bệnh của E. coli với tiêu chảy ở lợn con, cho thấy, chủng E. coli ETEC phân lập được có tỷ lệ khác nhau về năm tổ hợp yếu tố gây bệnh là LT+STa+K88+Hly+ (52,2%), LT+STa+STb +K88+Hly-(11,1%), STa+K99+(17,2%), STa+ STb+(3,3%) và STb (15,7%).…”

Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

“…This idea was suggested by Dorfman in 1943 (6) and has been used for the serological diagnosis of infectious diseases. Pooling samples is also an efficient way to screen for the nucleic acids of viruses, bacteria, or parasites (4,5,(7)(8)(9)(10)(11)(12)(13)(14)(15). However, pooling can decrease the sensitivity of assays due to the dilution of the samples, which is problematic in clinical diagnostics (16).…”

“…To prevent a loss of sensitivity, it may also be necessary to limit pool sizes to limit sample dilutions (7,10,14) or to use special kits for extracting DNA from larger sample volumes (4, 16). With our strategy, it is possible to pool a larger number of specimens (up to 20) and to lyophilize DNA specimens to a volume of 1.6 ml, while commercial DNA extraction kits are limited to 1 ml (4,16).…”