Ammonia oxidation is known to be carried out by ammonia-oxidizing bacteria (AOB) and archaea (AOA), while methanotrophs (methane-oxidizing bacteria (MOB)) play an important role in mitigating methane emissions from the environment. However, the difference of AOA, AOB, and MOB distribution in wetland sediment and adjacent upland soil remains unclear. The present study investigated the abundances and community structures of AOA, AOB, and MOB in sediments of a high-altitude freshwater wetland in Yunnan Province (China) and adjacent agricultural soils. Variations of AOA, AOB, and MOB community sizes and structures were found in water lily-vegetated and Acorus calamus-vegetated sediments and agricultural soils (unflooded rice soil, cabbage soil, and garlic soil and flooded rice soil). AOB community size was higher than AOA in agricultural soils and lily-vegetated sediment, but lower in A. calamus-vegetated sediment. MOB showed a much higher abundance than AOA and AOB. Flooded rice soil had the largest AOA, AOB, and MOB community sizes. Principal coordinate analyses and Jackknife Environment Clusters analyses suggested that unflooded and flooded rice soils had relatively similar AOA, AOB, and MOB structures. Cabbage soil and A. calamus-vegetated sediment had relatively similar AOA and AOB structures, but their MOB structures showed a large difference. Nitrososphaera-like microorganisms were the predominant AOA species in garlic soil but were present with a low abundance in unflooded rice soil and cabbage soil. Nitrosospira-like AOB were dominant in wetland sediments and agricultural soils. Type I MOB Methylocaldum and type II MOB Methylocystis were dominant in wetland sediments and agricultural soils. Moreover, Pearson's correlation analysis indicated that AOA Shannon diversity was positively correlated with the ratio of organic carbon to nitrogen (p < 0.05). This work could provide some new insights toward ammonia and methane oxidation in soil and wetland sediment ecosystems.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the singlestranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from “samples to results” within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

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