FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Molecular epidemiology of COVID-19 epidemic in Ningxia, 2021

Objective: To understand the molecular epidemiological characteristics of COVID-19 in Ningxia, and provide evidence for the surveillance, prevention and control of COVID-19.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

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 single-stranded 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 ß 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.

Visualized Genotyping from "Sample to Results" Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

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.

A systematic review of etiology, epidemiology, clinical manifestations, image findings, and medication of 2019 Corona Virus Disease-19 in Wuhan, China.

BACKGROUND: Unknown origin pneumonia has been furiously spreading since the late of December 2019, subsequently spread to approximately all provinces and areas in China and many countries, which was announced as a Public Health Emergency of International Concern by World Health Organization (WHO). The studies on 2019 Corona Virus Disease-19 (COVID-19) conducted from various fields around the world. Herein, the objective of the present study is to summarize the etiology, epidemiology, clinical manifestations, image findings, traceability analysis, and drug development of COVID-19. METHODS: The following electronic databases were searched: Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database, VIP Chinese Science and Technology Periodical Database, and Wanfang Data. Other relevant literature will be manually searched as a compliment. We have reviewed etiology, epidemiology, clinical manifestations, image findings, and medication from case reports and retrospective clinical studies relating to COVID-19 published since the outbreak. RESULTS: The coronavirus is closely related to bat coronavirus and pangolin coronavirus. Besides, the infection pathway is confirmed to be the respiratory and digestive systems. The virus indicates person-to-person transmission and some patients present asymptomatic. The elderly have a higher mortality rate. Rapid and sensitive nucleic acid testing is usually used as a basis for diagnosis. Currently, there is no specific vaccine and antiviral drug. Intervention actions such as travel bans and quarantine adopted have effectively reduced the spread of the epidemic. CONCLUSION: This systemic review will provide high-quality evidence to summarize etiology, epidemiology, clinical manifestations, image findings, traceability analysis, drug development in patients with COVID-19.