Comparison data of a two-target real-time PCR assay with and without an internal control in detecting Salmonella enterica from cattle lymph nodes

Abstract

A real-time PCR (qPCR) assay targeting on invA and pagC genes was developed and validated for the detection and quantification of Salmonella enterica strains (Bai et al., 2018) [1]. A host gene, normally an endogenous housekeeping gene (Beer-Davidson et al., 2018; Poon et al., 2004) [2,3], or an irrelevant exogenous gene (Cheng et al., 2015; Sedlak et al., 2014) [4,5] has been widely used as an internal control to monitor nucleic acid extraction efficiencies and potential PCR inhibitions in PCR-based detection assays. An endogenous internal control designed based on the 18S rRNA gene was used in the above-mentioned qPCR assay. This 18S rRNA internal control amplifies the target gene in multiple species including bovine, swine, ovine, caprine and cervine. Data was generated by the duplex qPCR assay on 138 enriched cattle lymph node samples without the internal control, and compared with data on the same samples tested by the triplex qPCR assay that has the 18S rRNA gene as internal control. Threshold cycle (Ct) data for the duplex and the triplex qPCR on the 138 samples were similar, and are presented in this brief report.

Evaluation of a Porcine Endogenous Reference Gene (Internal Sample Control) in a Porcine Reproductive and Respiratory Syndrome Virus RT-qPCR

“…Housekeeping genes have been used as ISCs in human [ 24 , 25 , 26 ] and veterinary diagnostic research [ 6 , 10 , 11 ] because they are essential to basic cellular functions and are, therefore, innate to the biological specimens being tested [ 27 ]. For that reason, failure to detect the ISC would indicate a fault at some point between sample collection and final PCR testing.…”

“…Housekeeping genes frequently used as ISCs include glyceraldehyde 3-phosphate dehydrogenase, β-actin, 18S rRNA, peptidyl-prolyl cis-trans isomerase, β-2-microglobulin, and ubiquitin C [ 8 , 9 ]. Although not widely investigated, the use of ISCs in veterinary diagnostic has been reported for several species, e.g., 18S rRNA for the DNA detection of Salmonella enterica in cattle lymph nodes [ 10 ], bird β-actin for the RT-qPCR detection of avian influenza virus [ 6 ], and porcine β-actin for the detection of African swine fever virus by qPCR [ 11 ].…”

Effect of extrinsic factors on the detection of PRRSV and a porcine-specific internal sample control in serum, oral fluid, and fecal specimens tested by RT-rtPCR

“…12 Research in veterinary laboratory medicine has reported the use of species-specific ISCs in an avian influenza A virus reverse-transcription rtPCR (RT-rtPCR) assay (β-actin gene), 36 African swine fever virus rtPCR assay (β-actin gene), 37 and a PCR assay for the detection of Salmonella enterica in cattle. 3 Initially identified in 1991, porcine reproductive and respiratory syndrome virus (PRRSV; Betaarterivirus suid 1 and 2) continues to impose major costs on the global swine industry. 17,25,35 Strategies for PRRSV prevention and control invariably include diagnostic and surveillance testing to establish the infection status of individual pigs and/or groups of animals.…”

“…12 Research in veterinary laboratory medicine has reported the use of species-specific ISCs in an avian influenza A virus reverse-transcription rtPCR (RT-rtPCR) assay (β-actin gene), 36 African swine fever virus rtPCR assay (β-actin gene), 37 and a PCR assay for the detection of Salmonella enterica in cattle. 3…”