A novel strategy for the detection of SARS-CoV-2 variants based on multiplex PCR-MALDI-TOF MS

BackgroundThe second wave of coronavirus disease 2019 (COVID-19) has been incessantly causing catastrophe worldwide, and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants causes further uncertainty regarding epidemic risk. Here, a novel strategy for the detection of SARS-CoV-2 variants using multiplex PCR coupled with MALDI-TOF MS was developed. MethodsPlasmids carrying gene sequences containing 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants were synthesized. Using the nucleic acid sequence of SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2-variant-carrying plasmid, a MALDI-TOF MS method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect the above nine mutation types. The detection limit of this method was determined via the concentration gradient method. Twenty-one respiratory tract pathogens (9 bacteria, 11 respiratory viruses) and pharyngeal swab nucleic acid samples from healthy people were selected for specific validation. Sixteen samples from COVID-19 patients were used to verify the accuracy of this method. ResultsThe 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and all six variants (B.1.1.7, B.1.351, B.1.429, B.1.526, P.1 and B.1.617) could be identified. The detection limit for all 9 sites was 1.5x103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR CT values less than 30 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. ConclusionsMultiplex PCR-MALDI-TOF MS provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. Key pointAn all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-MALDI-TOF MS system was developed. All of the SARS-CoV-2 variants can be identified based on 9 types of 7 mutated sites of RBD of spike protein using this method.

Inhibitor screening of Spike variants reveals the heterogeneity of neutralizing antibodies to COVID-19 infection and vaccination

Mutations of the coronavirus responsible for coronavirus disease 2019 (COVID-19) could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronaviruss Spike (S) protein simultaneously. By screening purified antibodies and serum from convalescent COVID-19 patients and vaccinees against 72 S variants with the mSAIS assay, we identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n=104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and four prevalent S variants (D614G, B.1.1.7, B.1.351, P.1), thus demonstrating that high antibody diversity is associated with high NAb titers. Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development. HighlightsO_LIDeveloped a high throughput assay to screen the neutralizing effect of antibodies across multiple SARS-CoV-2 Spike variants simultaneously. C_LIO_LICharacterized the heterogeneity of neutralizing antibodies produced in response to COVID-19 infection and vaccination. C_LIO_LIDemonstrated the capacity of Spike variants neutralization is associated with the diversity of anti-Spike antibodies. C_LI

Transforming bacterial disease surveillance and investigation using whole-genome sequence to probe the trace / 医学前沿

Two decades have passed since the first bacterial whole-genome sequencing, which provides new opportunity for microbial genome. Consequently, considerable genetic diversity encoded by bacterial genomes and among the strains in the same species has been revealed. In recent years, genome sequencing techniques and bioinformatics have developed rapidly, which has resulted in transformation and expedited the application of strategy and methodology for bacterial genome comparison used in dissection of infectious disease epidemics. Bacterial whole-genome sequencing and bioinformatic computing allow genotyping to satisfy the requirements of epidemiological study in disease control. In this review, we outline the significance and summarize the roles of bacterial genome sequencing in the context of bacterial disease control and prevention.We discuss the applications of bacterial genome sequencing in outbreak detection, source tracing, transmission mode discovery, and new epidemic clone identification. Wide applications of genome sequencing and data sharing in infectious disease surveillance networks will considerably promote outbreak detection and early warning to prevent the dissemination of bacterial diseases.

Molecular epidemiology of carbapenem resistant Klebsiella pneumoniae in a hospital in Beijing / 中华预防医学杂志

Objective@#To reveal the molecular epidemiological characteristics of Carbapenem-Resistant Klebsiella pneumoniae (CRKP) isolated from a three level teaching hospital in Beijing.@*Methods@#Pulsed field gel electrophoresis (PFGE) was carried out to subtyping 375 CRKP isolated in that hospital between May 2010 and October 2015. Fifteen strains were chose based on the PFGE patterns to be analyzed by multi-locus sequence typing (MLST) and detection of carbapenem-resistance genes. One strain (A1502) was selected for whole genome sequencing and analyzing.@*Results@#The 375 CRKP were divided into 140 PFGE types, among which five types contained more than five strains. The dominant types were distributed in different time periods and wards. Among the 15 strains tested by MLST and carbapenem-resistance genes detection, 13 were ST11 strains carrying KPC-2 gene. By genome-based typing, A1502 was clustered together with strains from other hospitals of Beijing but far from the strains from Shanghai and Hangzhou.@*Conclusion@#The CRKP epidemic clone (ST11 clone carrying KPC-2) has been spreading within single hospital and across different hospitals in Beijing.

Progress in research of detection assay for pathogens causing community acquirerd pneumonia / 中华流行病学杂志

Community-acquired pneumonia (CAP) is a common respiratory infectious disease.The etiologic diagnosis of CAP remains an uneasy task.Early etiologic diagnosis is critical for proper treatment and might improve the prognosis.So,it is important to identify pathogens causing CAP in early time and accurate way with sensitive and effective method.This paper summarizes the recent progress in the research of the detection assay for CAP.

Progress in research of detection assay for pathogens causing community acquirerd pneumonia / 中华流行病学杂志

Community-acquired pneumonia (CAP) is a common respiratory infectious disease.The etiologic diagnosis of CAP remains an uneasy task.Early etiologic diagnosis is critical for proper treatment and might improve the prognosis.So,it is important to identify pathogens causing CAP in early time and accurate way with sensitive and effective method.This paper summarizes the recent progress in the research of the detection assay for CAP.

Analysis of twin-arginine translocation system gene homology and transcription in Vibrio species / 中华预防医学杂志

<p><b>OBJECTIVE</b>To determine the function of twin-arginine translocation system (Tat) and gene cluster in Vibrio strains and to analyze the homology of tat gene cluster among different Vibrio spp. strains based on N16961 and tatABC mutant strains N169-dtat.</p><p><b>METHODS</b>Different serotypes of biotype strains of Vibrio spp. were selected to detect the transcription of 4 genes of Tat transport system and upstream ubi aarF gene and downstream cyt551 gene by the total RNA reverse transcription and homologicity of the gene cluster by sequencing analysis.</p><p><b>RESULTS</b>Our results showed that the 4 genes of tat cluster (tatA, tatB, tatC, and tatE) were intragenic and co-transcribed. We found that ubi aarF gene could be co-transcribed with tatA, tatB, but not with tatC. The electron transport chain and energy metabolism-related genes, cytochrome C551 peroxidase gene, and 4 genes located at upstream of tatABC operon were not transcribed with tatABC. Although the co-transcription between ubi aarF and tatAB was blocked in N169-dtat strain, they were still transcribed separately. Homologous analysis of genes of tat cluster in different types of Vibrio cholerae showed that tat gene cluster was a very conservative.</p><p><b>CONCLUSION</b>The ubi and aarF gene might be co-transcribed with genes of tat cluster in Vibrio cholerae, which and the close relationship showed that they might play a key function in Vibrio cholerae.</p>

The infection and molecular characteristics of Vibrio parahaemolyticus isolated from intestinal outpatient in two sentinel hospitals in Shanghai, 2010-2012 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To analyze virulence genes and molecular characteristics of Vibrio parahaemolyticus isolated from sporadic cases with diarrhea in tow sentinel hospitals of Shanghai, 2010-2012.</p><p><b>METHODS</b>A total of 2 729 stool samples were collected from two surveillance sentinel hospitals in Shanghai 2010-2012. Vibrio parahaemolyticus strains were isolated and identified from diarrhea out patients using TCBS agar plates and biochemical reactions. Thermostable direct hemolysingene (tdh), thermostable-related hemolysin gene (trh), hemolysin gene (tlh) were detected by multiplex PCR method. Isolates were analyzed by PFGE and MLST. The PFGE profiles were analyzed using BioNumerics software.</p><p><b>RESULTS</b>A total of 30 clinical Vibrio parahaemolyticus strains isolated from 2 729 stool samples. The anually Vibrio parahaemolyticus isolation rate during 2010 to 2012 were 1.1%(11/973), 1.0%(11/1 120) and 1.3%(8/636) respectively. The PCR positive rates of virulence genes tlh, tdh and trh were 100%, 97% and 0 respectively. The Vibrio parahaemolyticus strains were divided into 13 PFGE types (P1-P13)and 3 ST types (ST-189, ST-799, ST-3). Among 13 PFGE types, P4 was the main PFGE type, accounting for 30%(9/30). P9, P10 were accounting for 12% (4/30) respectively, P1, P2, P12, P13 were accounting for 7%(2/30) respectively, the others types were 3%(1/30) respectively. MLST analysis results showed there are three ST types, ST3 was 84%(25/30), ST189 and ST799 were accounting for 13% (4/30) and 3% (1/30) respectively.</p><p><b>CONCLUSION</b>The infection rate of Vibrio parahaemolyticus was not very high from 2010-2012 in Shanghai, all strains were positive for tlh and negative for trh. ST3 was the major type of Vibrio parahaemolyticus.</p>

Molecular characteristics and antibiotic resistance of Vibrio cholerae O139 in Shandong province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the molecular epidemiological characteristics and antibiotic resistance profiles of Vibrio cholerae O139 in Shandong province.</p><p><b>METHODS</b>A total of 13 strains of V. cholerae O139 (9 clinical strains and 4 environmental strains) isolated from cholera epidemics in Shandong province since 1997 were recovered and confirmed with serum agglutination and biochemical reaction. Pulsed-field gel electrophoresis (PFGE) was carried out for molecular subtyping. Virulence genes and drug resistance related genes were detected by PCR. Antibiotic susceptibility tests were performed using micro-broth dilution method.</p><p><b>RESULTS</b>Thirteen strains of V. cholerae O139 were differentiated into seven pulsetypes. One clinical strain and two environmental strains isolated from Jining in 2013 were clustered into the pulsetype namely KZGN11O139. CN0077, and an identical PFGE pattern of KZGN11O139. CN0002 was found among three clinical strains from Jinan in 2005, Jining in 2005 and Heze in 2009. Other pulsotypes were unique in China and found only in Shandong province. Because of deletion of ctxAB and tcpI, the PFGE patterns of two strains isolated from Yantai in 2000 and 2004 were different from other 11 strains which harbored ctxAB, tcpA, tcpI, rtxA, hlyA and toxR. All strains contained one or more drug resistance related genes such as intI 1, intI 4 and sxt, and were resistant to two kinds of antibiotics at least. Among the 12 kinds of antibiotics, the resistant ratioes to kamamycin, trimethoprim-sulfamethoxazole, ampicillin and gentamicin were 11/13, 9/13, 7/13 and 7/13, respectively.</p><p><b>CONCLUSION</b>Molecular subtyping indicates possible epidemiological links among V.cholerae O139 in Shandong province, and almost all strains were toxigenic and drug resistant.</p>