A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Tea plant-legume intercropping simultaneously improves soil fertility and tea quality by changing bacillus species composition.

Tea plant is an economically important crop in China, but long-term monoculture and substantial chemical nitrogen fertilizer input cause soil acidification, which in turn affects the nutrient supply and tea quality. Intercropping has drawn more attention in tea gardens because this pattern is expected to improve soil fertility and tea quality and change the soil microbial community composition. However, the roles of some key microorganisms in rhizosphere soils have not been well characterized. Hereby, a "soybean in summer and smooth vetch in winter" mode was selected to investigate the effects of intercropped legumes in a tea garden on soil fertility, tea quality, and the potential changes in beneficial bacteria such as Bacillus. Our data showed that when soybeans were turned into soil, intercropping system exhibited higher soil organic matter (SOM), total nitrogen (TN), tea quality indices and the expression of Camellia sinensis glutamine synthetase gene (CsGS). Notably, intercropping significantly affected the bacterial communities and decreased the relative abundance of Bacillus but increased its absolute abundance. Bacillus amyloliquefaciens BM1 was isolated from intercropped soil and showed outstanding plant growth-promoting (PGP) properties when coinoculated with rhizobia. In winter, intercropping with smooth vetch had a beneficial effect on soil properties and tea quality. Comparably, coinoculation with strain BM1 and Rhizobium leguminosarum Vic5 on smooth vetch (Vicia villosa) showed huge improvements in SOM, TN and quality of tea leaves, accompanied by the highest level of amino acids and lowest levels of polyphenol and caffeine (p < 0.05). According to these results, our findings demonstrate that intercropping with some legumes in the tea garden is a strategy that increases SOM, TN and tea quality, and some PGP Bacillus species are optional to obtain an amplification effect.