Standard curve for Listeria qPCR

<p dir="ltr">Abortions are one of the causes of major economic losses in both small- and large-scale livestock production systems and wildlife. The primary etiology of reproductive disorders in domesticated and wild ruminant animals is from infectious agents, and these include listeriosis caused by bacteria in the Listeria genus. As listeriosis is foodborne and causes zoonosis, it is indeed important to identify the causal agents where abortion is concerned. It will provide valuable information for the timely control of the disease. Isolation and identification of these bacteria has relied mainly on traditional culturing and serological techniques. These are, however, limited in accuracy, sensitivity, conclusiveness, and timeliness. This study aimed to develop and employ a polymerase chain reaction (PCR)-based technique in the detection of Listeria species in tissues of abortive products of ruminants from different provinces in South Africa. The qPCR was optimized by using reference bacterial isolates, namely L. monocytogenes and L. ivanovii, known to cause abortions in animals. DNA was extracted from these bacterial isolates and amplified using TaqMan-based qPCR chemistry. The optimal primer and probe concentrations were determined to be 100 nM and 250 nM, respectively. </p><p dir="ltr"><br></p><p dir="ltr">Additionally, the efficiency and specificity of the qPCR assay were also assessed. Other bacteria encountered in products of abortion, such as Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 13311), Ochrobactrum anthropi (ATCC 49687), Streptococcus agalactiae (ATCC 27956), Staphylococcus aureus (ATCC 25923), and Acholeplasma laidlawlii (NCTC 10116), were included as non-target control samples. Our study successfully differentiated all the target controls from the non-target controls. Following this, the qPCR assays were employed to analyse tissue samples from products of abortion where pathological lesions were suspected for Listeria infection. No Listeria species were detected in these samples. This finding was supported by previous bacteriology data for these samples. It was further recommended that additional primer and probe sets be employed in multiplex qPCR assays in the identification of a wider range of the Listeria species.</p>