Mitochondrial Multiplex Real-Time PCR as a Source Tracking Method in Fecal-Contaminated Effluents

Abstract

Multiplex real-time PCR amplifying fecal mitochondrial DNA (mtDNA) combined with rapid, crude DNA preparations are promising additions to surface water source tracking methods. Amplification of eukaryotic mitochondrial DNA identifies the fecal source directly and can be used in conjunction with other intestinal microbial methods to characterize effluents. Species-specific primers and dual-labeled probes for human, swine, and bovine NADH dehydrogenase subunit 5 (ND5) genes were created for multiplex real-time PCR in feces and effluent slurries. The linear range of the multiplex assay was 10(2)-10(7) mtDNA copies for human, bovine, and swine effluent in combination (equal volumes). PCR amplification efficiencies for bovine, human, and swine mtDNA when assayed in combination were 93, 107, and 92% respectively. Linear regression correlation coefficients (r2) were 0.99 for all standard curves except for human mtDNA in combination (r2 = 0.95). Multiplex amplification of bovine, human, and swine mtDNA (ND5) exhibited no cross-reactions between the effluents from three species of interest. Also, no cross-reactions were observed with effluents of other vertebrates: sheep, goat, horse, dog, cat, Canada goose, broiler, layer, turkey, and tilapia. Performed as a blind test, the PCR operator was able to correctly identify all but two effluent challenge samples (10/12 or 83% correct) with no false positives (22/22 or 100% correct). The multiplex assay had a tendency to detect the species of highest mtDNA concentration only. Better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly. Real-time PCR detection limit was calculated as 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent. Some carry-over mtDNA PCR signal from consumed beef, but not pork, was found in feces of human volunteers.

Cow’s Milk Contamination of Human Milk Purchased via the Internet

“…biosearchtech.com, Petaluma, CA). 14 The probes contained a 39 black-hole quencher and a 59 6-carboxyfluorescein (FAM) (human) or Quasar 670 (bovine) fluorophore. Reactions (25 mL) comprised 12.5 mL iQ supermix (BioRad, Hercules, CA), species-specific primers (400 nM each), a speciesspecific probe (100 nM), 5 mL of DNA, and DNAse/RNAse free water.…”

Decay of Bacterial Pathogens, Fecal Indicators, and Real-Time Quantitative PCR Genetic Markers in Manure-Amended Soils

“…Salmonella enterica serovar Typhimurium and E. coli O157:H7 are two examples of bacterial pathogens that may enter VBNC states (8, 62). In addition, host-associated qPCR genetic markers are available and are reported to discriminate between human, cattle, swine, and other animal sources (4,23,25,32,35,(43)(44)(45), providing fecal pollution source information that may lead to better-informed remedial actions to improve water quality.Several of the emerging FIB and host-associated qPCR assays target gDNA from obligate anaerobic bacteria, primarily Bacteroidales. These organisms offer improved host specificity for development of host-associated qPCR assays and comprise a large fraction of the bacterial community in feces.…”

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

“…However, differences between the annealing temperatures of the primers prevented the detection of the target gene and the exogenous internal control in a single temperature cycle. Nevertheless, we included the mitochondrial mRNA encoding bovine NADH-dehydrogenase-5 (Caldwell et al 2007) based on a favorable report by Asano et al (2010), who used this control to validate a PCR assay for the VP1 gene of RV, in agreement with our finding of accurate detection in Real-Time PCR, where C t values between 22.07 and 27.11 and low variability in T m (78.76±0.28°C) was obtained.…”

“…The samples were then tested by amplification of the messenger RNA (mRNA) of the gene encoding the mitochondrial bovine NADHdehydrogenase-5 protein using a pair of primers (BOV-F and BOV--R), described by Caldwell, Raley & Levine (2007), which generates a product of 191-bp. The following amplification conditions were used: 95°C for 10 min; 40 cycles of 94°C for 1 min, 55°C for 2min, and 72°C for 1 min, followed by a standard melting curve of the product (95°C for 15s, 60°C for 1 min, and 95°C for 15s with a reading at every 0.3°C).…”