Standard curve for Listeria qPCR

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. <br>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.

流产是中小型及大型畜牧生产系统与野生动物面临的重大经济损失诱因之一。家养与野生反刍动物生殖紊乱的主要病因是传染性病原体，其中包括李斯特菌属（Listeria）细菌引发的李斯特菌病。由于李斯特菌病属于食源性疾病且可导致人畜共患病，明确与流产相关的致病原具有重要意义，可为该疾病的及时防控提供宝贵信息。
此前对这类细菌的分离与鉴定主要依赖传统培养及血清学技术，但此类方法在准确性、灵敏度、结论可靠性与时效性方面均存在局限。本研究旨在开发并应用基于聚合酶链反应（PCR）的技术，对南非不同省份反刍动物流产产物组织中的李斯特菌属物种进行检测。
研究以已知可引发动物流产的单核细胞增生李斯特菌（L. monocytogenes）与伊万诺夫李斯特菌（L. ivanovii）作为参考细菌分离株，对定量聚合酶链反应（qPCR）进行优化。从上述细菌分离株中提取DNA，并采用基于TaqMan的qPCR化学体系进行扩增，最终确定最优引物与探针浓度分别为100 nM与250 nM。
此外，本研究还评估了该qPCR检测方法的效率与特异性。选取流产产物中常见的其他细菌作为非靶标对照样本，包括大肠埃希菌（Escherichia coli, ATCC 25922）、鼠伤寒沙门氏菌（Salmonella typhimurium, ATCC 13311）、人苍白杆菌（Ochrobactrum anthropi, ATCC 49687）、无乳链球菌（Streptococcus agalactiae, ATCC 27956）、金黄色葡萄球菌（Staphylococcus aureus, ATCC 25923）以及无胆甾原体（Acholeplasma laidlawlii, NCTC 10116）。本研究成功将所有靶标对照与非靶标对照区分开来。
随后，利用该qPCR检测方法对疑似存在李斯特菌感染病理病变的流产产物组织样本进行分析，未检出李斯特菌属物种。这一结果得到了对应样本既往细菌学检测数据的支持。研究最后建议，后续可采用多重qPCR检测体系搭配更多引物与探针组合，以实现对更广范围李斯特菌属物种的鉴定。