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 .
Cryptococcus neoformans ( C. neoformans )/ C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans / C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans / C. gattii . In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64 , of C. neoformans / C. gattii , and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/μL or 1 fg/μL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans / C. gattii , meeting the need for rapid, specific, and sensitive detection.
The cDNA fragment covering full-length sequence of RStV RNA3 of Yunnan isolate in China was obtained by RT-PCR. The PCR-derived fragment was then cloned into vector pCRII. The cloned cDNA was sequenced. Comparison of the nucleotide and deduced amino acid sequences with those of the Japanese isolate T was made. The results indicated that the Chinese isolate Y is closely related to the Japanese isolate T.
Abstract Background: Enterobacter cloacae exhibits strong adhesion and invasion properties which can contribute its ability to infect the host; it has been considered as an important opportunistic pathogen throughout the world. Simple, rapid, and accurate detection methods are needed to control the spread of E . cloacae. Current methods suffer from various shortcomings and do not meet the demand for on-site detection. Results: In this study, an isothermal detection method using recombinase polymerase amplification combined with lateral flow strip (RPA-LFS) was established to target the outer membrane protein X ( omp X) gene of E . cloacae. This reaction can be performed in 30 min at 37°C. The limit of detection of 10 1 CFU/reaction was equivalent to that of the qPCR method. The detection accuracy of clinical samples was also equal to that of the qPCR method. Conclusions: The RPA-LFS assay developed in this study was simple, rapid, and accurate, and did not require a laboratory facility. It may be useful for on-site detection of E . cloacae.
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