An overview of calf diarrhea - infectious etiology, diagnosis, and intervention

Cho, Yong-il; Yoon, Kyoung-Jin

Polymerase Chain Reaction (PCR) is a common nucleic acid-based method for detecting enteric pathogens. PCR involves thermocyclic enzymatic amplification of specific DNA sequences of the target pathogen using a pair of oligonucleotide primers that hybridize to DNA/cDNA regions of interest in the genomic sequence. Genomic material of the target pathogen is first extracted. Next, the sample is mixed with a heat-stable DNA polymerase (e.g., Taq DNA polymerase), dNTPs, primers, and PCR buffer. DNA amplification usually proceeds for 25 to 40 cycles in an automated thermal cycler [30] . Each cycle includes a double-stranded DNA denaturation step, primer annealing to each DNA strand, and polymerization of a new strand. After completion of the reaction, the PCR products can be visualized on an agarose or acrylamide gel after electrophoresis and staining with ethidium bromide that binds to double-stranded DNA. Successful amplification of the target sequence is determined based on molecular size and/or sequencing of the PCR product. PCR testing is especially useful for detecting viruses that are difficult to isolate in cell culture or bacteria that require a long time to grow [31] . There are numerous commercial PCR reagents available which provide convenience, high sensitivity, and rapid results. PCR testing requires trained and experienced technicians. Inadvertent contamination during sampling in the field or processing at the laboratory can be a source of false positive results due to its high sensitivity. Viruses with a high mutation rate, often RNA viruses (e.g., rotavirus and calicivirus), need to be continuously monitored for sequence changes in the target gene otherwise negative results will be obtained due to primer incompatibility. Fecal samples are known to contain factors that inhibit PCR and can lead to false negative results if appropriate reagents or steps to remove such inhibitory substances are not included in the test procedures.