Zhang Shuxin, Shi Jiahua, Li Xuan, Coin Lachlan, O'Brien Jake W, Sivakumar Muttucumaru, Hai Faisal, Jiang Guangming
School of Civil, Mining and Environmental Engineering, University of Wollongong, Australia.
Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, Australia; School of Medical, Indigenous and Health Sciences, University of Wollongong, Australia.
Sci Total Environ. 2023 Sep 20;892:164574. doi: 10.1016/j.scitotenv.2023.164574. Epub 2023 Jun 1.
Campylobacter spp. is one of the most frequent pathogens of bacterial gastroenteritis recorded worldwide. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the two major disease-associated species, accounting for >95 % of infections, and thus have been selected for disease surveillance. Monitoring temporal variations in pathogen concentration and diversity excreted from community wastewater allows the early detection of outbreaks. Multiplex real-time/quantitative PCR (qPCR) enables multi-target quantification of pathogens in various types of samples including wastewater. Also, an internal amplification control (IAC) is required for each sample when adopting PCR-based methods for pathogen detection and quantification in wastewater to exclude the inhibition of the wastewater matrix. To achieve reliable quantification of C. jejuni and C. coli towards wastewater samples, this study developed and optimized a triplex qPCR assay by combining three qPCR primer-probe sets targeting Campylobacter jejuni subsp. jejuni, Campylobacter coli, and Campylobacter sputorum biovar sputorum (C. sputorum), respectively. This triplex qPCR assay not only can directly and simultaneously detect the concentration of C. jejuni and C. coli in wastewater but also can achieve the PCR inhibition control using C. sputorum primer-probe set. This is the first developed triplex qPCR assay with IAC for C. jejuni and C. coli, to be used in the wastewater-based epidemiology (WBE) applications. The optimized triplex qPCR assay enables the detection limit of the assay (ALOD and wastewater (PLOD) as 10 gene copy/μL and 2 log10 cells/mL (2 gene copies/μL of extracted DNA), respectively. The application of this triplex qPCR to 52 real raw wastewater samples from 13 wastewater treatment plants demonstrated its potential as a high-throughput and economically viable tool for the long-term monitoring of C. jejuni and C. coli prevalence in communities and the surrounding environments. This study provided an accessible methodology and a solid foundation for WBE-based monitoring of Campylobacter spp. relevant diseases and paved the road for future WBE back-estimation of C. jejuni and C. coli prevalence.
弯曲杆菌属是全球记录的细菌性肠胃炎最常见的病原体之一。空肠弯曲杆菌（C. jejuni）和结肠弯曲杆菌（C. coli）是两种主要的与疾病相关的菌种，占感染病例的95%以上，因此被选作疾病监测对象。监测社区废水中排出的病原体浓度和多样性的时间变化有助于早期发现疫情爆发。多重实时/定量PCR（qPCR）能够对包括废水在内的各种类型样本中的病原体进行多靶点定量分析。此外，在采用基于PCR的方法检测和定量废水中的病原体时，每个样本都需要一个内部扩增对照（IAC），以排除废水基质的抑制作用。为了实现对废水中空肠弯曲杆菌和结肠弯曲杆菌的可靠定量分析，本研究通过组合分别靶向空肠弯曲杆菌空肠亚种、结肠弯曲杆菌和唾液弯曲杆菌生物变种（C. sputorum）的三个qPCR引物-探针组，开发并优化了一种三重qPCR检测方法。这种三重qPCR检测方法不仅可以直接同时检测废水中空肠弯曲杆菌和结肠弯曲杆菌的浓度，还可以使用唾液弯曲杆菌引物-探针组实现PCR抑制控制。这是首个开发的用于空肠弯曲杆菌和结肠弯曲杆菌的带有IAC的三重qPCR检测方法，可用于基于废水的流行病学（WBE）应用。优化后的三重qPCR检测方法使该检测方法的检测限（ALOD）和废水中的检测限（PLOD）分别为10个基因拷贝/μL和2 log10个细胞/mL（提取DNA为2个基因拷贝/μL）。将这种三重qPCR应用于来自13个污水处理厂的52个实际原废水样本，证明了其作为一种高通量且经济可行的工具，用于长期监测社区及周边环境中空肠弯曲杆菌和结肠弯曲杆菌流行情况的潜力。本研究为基于WBE的弯曲杆菌属相关疾病监测提供了一种可及的方法和坚实基础，并为未来空肠弯曲杆菌和结肠弯曲杆菌流行情况的WBE反向估计铺平了道路。
