Multiplex polymerase chain reaction to detect Salmonella serovars Indiana, Enteritidis, and Typhimurium in raw meat

Abstract

Salmonella‐contaminated raw food is an important source of human food poisoning. Traditional methods for detecting Salmonella in meat involve many time‐consuming and laborious steps, preventing timely and effective control of the pathogen. In this study, a multiplex polymerase chain reaction (PCR) assay that targets serovar‐specific genes such as A7P63_0910 (Salmonella Indiana), sdfI (Salmonella Enteritidis), and STM4497 (Salmonella Typhimurium) for three meat‐associated Salmonella serovars was established. This multiplex PCR has been validated by extensive testing on reference strains and clinical isolates belonging to different Salmonella serovars (n = 348) and non‐Salmonella bacteria (n = 12). The detection limit is 100 CFU per reaction for bacterial culture, being equivalent to 10 pg per reaction for bacterial genomic DNA. Detection of Salmonella in raw meat samples by this multiplex PCR revealed that the prevalence of S. Indiana, S. Enteritidis, and S. Typhimurium is 7.7%, 5.7%, and 2.0%, respectively. The multiplex PCR established in this study can be used as a novel and convenient tool for simultaneous detection of three major meat‐associated Salmonella serovars, including the newly emerging and highly drug‐resistant S. Indiana. Practical applications In this study, a multiplex polymerase chain reaction (PCR) assay that targets serovar‐specific genes such as A7P63_0910 (Salmonella Indiana), sdfI (Salmonella Enteritidis), and STM4497 (Salmonella Typhimurium) for three meat‐associated Salmonella serovars was established. This multiplex PCR is suitable for the simultaneous detection and differentiation of three major meat‐associated Salmonella serovars, and has been validated by extensive testing of reference strains and clinical isolates. The multiplex PCR assay can provide rapid and specific screening for top three Salmonella serovars in meat samples.

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

“…Therefore, there was no sophisticated instruments or requirement for specialist technicians. Compared with our previous publication [ 23 ], our present reaction can be performed on a simple heat block and carried out by an operator with basic training. Our assay is simpler and more affordable than that of PCR-based methods, making it suitable for resource-limited settings.…”

“…Here, the A7P63_0910 (GenBank: ANF77768.1) gene was chosen as the specific target for S. Indiana, which is uniquely present in S. Indiana, but not in other Salmonella serovars or any non- Salmonella bacteria, and used as a target gene for S. Indiana detection [ 23 ]. A total of 7 pairs of RPA primers (named SI-1 to SI-7, seen in Table 2 ) were designed according to the conserved region of A7P63_0910, and the amplification products were analyzed by electrophoresis in agarose gels.…”

An Aggregation-Induced Emission Material Labeling Antigen-Based Lateral Flow Immunoassay Strip for Rapid Detection of Escherichia coli O157:H7

“…It is not suitable for the detection of a large number of samples and cannot monitor the epidemic in real time. 5 Methods based on molecular biology include polymerase chain reaction (PCR), 6 real-time PCR, 7 multiplex PCR, 8 loop-mediated isothermal amplification (LAMP), 9 rolling circle amplification (RCA), 10 and recombinase polymerase amplification (RPA). 11 Although molecular biology methods have high sensitivity, they require specialized equipment and skilled technicians to carry out complex operation procedures.…”

Rapid detection of Escherichia coli O157:H7 in milk, bread, and jelly by lac dye coloration‐based bidirectional lateral flow immunoassay strip

“…The detection methods for foodborne pathogens were based on molecular biology and immunology. The molecular biology‐based methods incorporate polymerase chain reaction (PCR) (Hull‐Jackson, Mota‐Meira, & Adesiyun, 2019), real‐time PCR (Alia, Andrade, Cordoba, Martin, & Rodriguez, 2020; Zhong, Tian, Wang, & Wang, 2016), multiplex PCR (Chen, Tang, Liu, Cai, & Bai, 2012; Wei et al, 2019; Xu et al, 2019), loop‐mediated isothermal amplification (LAMP) (Arunrut, Kiatpathomchai, & Ananchaipattana, 2018; Kampeera et al, 2019; Wan, Lu, Bie, Lv, & Zhao, 2020), recombinase polymerase amplification (RPA) (Ren et al, 2020), and rolling circle amplification (RCA) (Jiang et al, 2019). Although these methods can achieve high sensitivity, the requirement of high‐quality professional technicians and instruments limit the use in special places.…”