Dataset Information

Rapid and specific detection of Enterococcus faecalis with a visualized isothermal amplification method.

ABSTRACT: Enterococcus faecalis is a serious problem for hospitals and can spread from patient to patient. Most of the current detection methods are associated with limitations associated with the need for trained personnel; they are also time-consuming. Thus, it is necessary to develop rapid and accurate detection methods to control the spread of E. faecalis. In this study, we developed a rapid and accurate detection method for E. faecalis using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS). This method could be completed in approximately 35 min at 37°C. The limit of detection was 10 CFU/µL, irrespective of whether the templates were pure or complex. This method also showed good specificity and compatibility. In total, 278 clinical samples were tested using the RPA-LFS method; the detection accuracy was equal to that of the conventional qPCR method. This visualized isothermal amplification method could be useful for the future on-site detection of E. faecalis.

SUBMITTER: Zhu B

PROVIDER: S-EPMC9510690 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid and specific detection of Enterococcus faecalis with a visualized isothermal amplification method.

Zhu Bo B Hu Juan J Li Xuelian X Li Xiaomin X Wang Lei L Fan Shihui S Jin Xin X Wang Kun K Zhao Weiguo W Zhu Wenjun W Chen Cheng C Wang Zilu Z Lu Yingzhi Y

Frontiers in cellular and infection microbiology 20220912

Enterococcus faecalis is a serious problem for hospitals and can spread from patient to patient. Most of the current detection methods are associated with limitations associated with the need for trained personnel; they are also time-consuming. Thus, it is necessary to develop rapid and accurate detection methods to control the spread of E. faecalis. In this study, we developed a rapid and accurate detection method for E. faecalis using recombinase polymerase amplification ( ...[more]

PMID: 36171761

Similar Datasets

Project description:BACKGROUND:Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. RESULTS:A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170 fg of genomic DNA, or 6.25 × 103 CFU/25 g in spiked shrimp without any enrichment. The method finishes detection within 30 min at temperatures between 35 °C and 45 °C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected. CONCLUSIONS:The RPA-LFD method developed in this study is fast, simple, highly specific and does not require complicated equipment. This method is applicable for on-site detection of V. alginolyticus pathogenic strains for the mariculture industry.

Project description:Vibrio cholerae is a leading waterborne pathogenic bacterium worldwide. It can cause human cholera that is still pandemic in developing nations. Detection of V. cholerae contamination in drinking water and aquatic products is imperative for assuring food safety. In this study, a simple, sensitive, specific, and visualized method was developed based on loop-mediated isothermal amplification (LAMP) (designated sssvLAMP) to detect virulence-associated (ctxA, tcpA, hapA, mshA, pilA, and tlh) and species-specific (lolB) genes of V. cholerae. Three pairs of oligonucleotide primers (inner, outer, and loop primers) were designed and or synthesized to target each of these genes. The optimal conditions of the sssvLAMP method was determined, and one-step sssvLAMP reaction was performed at 65°C for 40 min. Positive results were simply read by the naked eye via color change (from orange to light green) under the visible light, or by the production of green fluorescence under the UV light (260 nm). The sssvLAMP method was more efficient in detecting 6.50 × 101-6.45 × 104-fold low number of V. cholerae cells, and more sensitive in V. cholerae genomic DNA (1.36 × 10-2-4.42 × 10-6 ng/reaction) than polymerase chain reaction (PCR) method. Among 52 strains of V. cholerae and 50 strains of non-target species (e.g., other Vibrios and common pathogens) examined, the sensitivity and specificity of the sssvLAMP method were 100% for all the target genes. Similar high efficiency of the method was observed when tested with spiked samples of water and aquatic products, as well as human stool specimens. Water from various sources and commonly consumed fish samples were promptly screened by this simple and efficient visualized method and diversified variation in the occurrence of the target genes was observed. V. cholerae strains could be mostly detected by the presence of hapA and tlh alone or in combination with other genes, indicating a variable risk of potentially pathogenic non-O1/O139 strains in edible food products. This novel LAMP method can be a promising tool to address the increasing need of food safety control of aquatic products.

Dataset Information

Rapid and specific detection of Enterococcus faecalis with a visualized isothermal amplification method.

Publications

Rapid and specific detection of <i>Enterococcus faecalis</i> with a visualized isothermal amplification method.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets