ABSTRACT
Objective To establish and evaluate a method for rapid and sensitive S.xylosus detection using qPCR(real-time quantitative PCR).Methods A gehM gene fragment was selected as the target for S.xylosus.A set of specific primers was synthesized and a qPCR method was established to detect S.xylosus.A S.xylosus standard strain and other non-target strains were chosen for analysis.DNA of S.xylosus was diluted 10-fold to determine its sensitivity.Clinical samples were tested,and positive products were sequenced.The result were compared with those of bacterial culture.Results S.xylosus had a specific amplification curve,whereas other non-S.xylosus species did not,indicating that the primers were specific for S.xylosus.Sensitivity was 100 fg/μL DNA.Repeatability within and between groups was less than 3％.A total of 60 clinical samples were analyzed,of which five samples had a typical S curve.qPCR products were sequenced and BLAST searched.The similarity of the gene sequences was 99.63％,indicating that the sample was positive for the S.xylosus gehM gene with a positivity rate of 8.3％.However,the positivity rate of bacterial culture was 6.7％.The positivity rate of qPCR was slightly higher than that of the culture.Conclusions The established qPCR method is rapid with high sensitivity and specificity,and can be used to detect S.xylosus.
ABSTRACT
The microbiological quality of laboratory animals is crucial for the validity and reproducibility of scientific research data, as well as human health and animal welfare. Currently, individual ventilation cages (IVC) have become the mainstream feeding system for rodent laboratory animals. The most commonly used pathogen monitoring method for this feeding system is soiled bedding sentinels (SBS). This method monitors the microbial carrying status of mouse colony through indirect contact and delayed feedback. It can effectively monitor pathogens transmitted via the fecal-oral route, such as mouse hepatitis virus and reovirus. However, this method has difficulty detecting pathogens mainly transmitted through aerosols or direct contact, such as Sendai virus and Pasteurella pneumotropica. The exhaust air dust (EAD)-PCR monitoring method involves swab sampling in the IVC exhaust ducts to monitor the corresponding racks of the ducts; swab sampling before the prefiltration of the host to monitor the entire IVC rack; and EAD collection device sampling to monitor all racks connected to the same host. Different IVC manufacturers have developed corresponding EAD collection devices for their respective IVC systems, making operations convenient and standardization easy. Compared with the SBS method, the EAD-PCR method significantly improves detection rate and timeliness, with the fastest detection possible after one week of exposure. It can serve as a supplement or replacement for the SBS method. Currently, increasing evidence supports that EAD-PCR testing is a more reliable, sensitive, and cost-effective monitoring method, and is more beneficial to animal welfare. This article reviews the application progress of these two methods for monitoring pathogens, analyzes the existing limitations of the EAD-PCR method, and proposes solutions based on its implementation in our laboratory and examination units. The EAD-PCR method helps reduce the number of live sentinel animals used in pathogen monitoring, in order to better maintain the "3Rs" principle of laboratory animal welfare.