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OBJECTIVE@#To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.@*METHODS@#We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.@*RESULTS@#This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.@*CONCLUSION@#By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
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Humans , COVID-19 , CRISPR-Cas Systems , Genotype , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , RNA , COVID-19 TestingABSTRACT
Objective:To obtain the genome sequences of SARS-CoV-2 in respiratory specimens in Guangdong Province with next-generation sequencing (NGS) and analyze the factors influencing sequencing.Methods:Eight upper and lower respiratory tract specimens were collected from patients with SARS-CoV-2 infection in Guangdong Province in January 2020. RNA library construction was used to obtain the genome sequences of SARS-CoV-2. A bio-informatics software package (CLC Genomics Workbench 12.0) was used to analyze and compare the genomic sequences.Results:Five SARS-CoV-2 genome sequences were obtained from the eight specimens and two were obtained from lower respiratory tract specimens. The nucleotide homology to SARS-CoV-2 was 97.74%-99.90%. The Ct values were lower, while the sequencing depth, coverage, relative abundance and genome integrity were higher in sequencing the SARS-CoV-2 in lower respiratory tract specimens.Conclusions:The low Ct value of SARS-CoV-2 in the samples was good for sequencing.
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Objective To detect the positive rates of antibodies against avian influenza virus (AIV) subtypes H5, H6, H7 and H9 among people in poultry occupations in Guangdong province and to analyze the transmission of various subtypes of AIV from poultry to human contacts for the prevention and control of novel AIV infection in human beings.Methods Serum specimens were collected from 1066 peo-ple in poultry occupations ( occupational group) and 205 people not in poultry occupations ( non-occupational group) in 10 cities of Guangdong province.The inactivated AIV strains, isolated from poultry or environment of Guangdong province, were used as antigens to detect antibodies against AIV subtypes H5, H6, H7 and H9 by using the hemagglutination inhibition ( HI) assay.Results The positive rates of antibodies against AIV subtypes H5, H6, H7 and H9 carried by people from the occupational group were respectively 0.44%, 0%, 0.30%and 0.30%in 2013 and 1.08%, 0.0%, 0.0%and 0.27%in 2014.Only the anti-H9 anti-bodies were detected in serum samples collected form people in the non-occupational group in 2013 with a positive rate of 0.95%.No significant differences with the positive rates of anti-AIV antibodies were found between the occupational group and the non-occupational group.However, the geometric mean titer ( GMT) of anti-AVI antibodies in people from the occupational group was higher than that of the non-occupational group.Conclusion Although a grand spread of AIV from avian to human is not likely to happen yet, con-tacting with poultry is the risk factor for AIV infection in Guangdong population.A long-term surveillance of anti-AIV antibodies in serum should be strengthened among people in poultry occupations for the timely pre-vention and control of novel AIV outbreak.
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To investigate the genetic character and origin of the first imported infection case of middle East respiratory syndrome coronavirus (named as MERS-CoV_China GD01), RNA was extracted from swabs of this patient followed by RT-PCR amplification. All coding gene of structural (S, E, M, E) and accessory (ORF3, ORF4a, ORF4b, ORF5, ORF8b) proteins were sequenced and analyzed. Phylogenetic analyses of structural protein coding genes of MERS-CoV_ China GD01 indicates that several substitutes exists in S coding gene and its origin belong group 5 of MERS-CoV, which were recent circulated in Saudi Arabia area, while other three structural genes (N, E, M) were very conserved. Phylogenetic analyses of accessory protein coding genes of MERS-CoV China GD01 indicates that several substitutes exists among ORF3, ORF4a, ORF4b and ORF5, while ORF8b was conserved. In conclusion, genome of MERS-CoV_ China GD01 was general conserved although several genetic variations were found among structural and accessory protein coding genes. This is the first report on sequencing and phylogenetic analyses of the first imported MERS case in China, which may pay the way for prevention and control of imported MERS-CoV infection.
Subject(s)
Humans , China , Conserved Sequence , Coronavirus Infections , Virology , Evolution, Molecular , Genomics , Middle East Respiratory Syndrome Coronavirus , Genetics , Physiology , Phylogeny , Sequence Analysis , Viral Proteins , GeneticsABSTRACT
Objective To investigate the susceptibility to oseltamivir of influenza pandemic A (H1N1) 2009 viruses in Guangdong province during 2009,provide valuable information of prescription for clinics,and elucidate the variant trend of the epidemic strain based on phylogenetic analysis.Methods During April to December 2009,clinical specimens were collected from sentinel hospitals covering the whole Guangdong province.Virus isolation was performed by in MDCK cells or embryonated chicken eggs.A fluorescence-based neuraminidase (NA) enzyme inhibition assay was conducted to measure influenza susceptibility.The NAI susceptibility of influenza virus was expressed as the concentration of NAI needed to inhibit the NA enzyme activity by 50％.A subset of 68 viruses were performed NA sequencing for detecting resistant mutations and studying variant trends.Results During surveillance,221 influenza pandemic A ( H1N1 ) viruses were isolated.All strains were sensitive to oseltamivir inhibition assay,with a median IC50 of 0.24 nmol/L (range 0.02 -1.66 nmol/L).No mutation related to resistance was found.Phylogenic analysis illustrated that these NA genes were homology high to 99.5％ - 100.0％ with those from other countries.Conclusions influenza pandemic A (H1N1) 2009 viruses were sensitive to oseltamivir in Guangdong,and useful for prophylaxis and treatment of influenza infection.Little selective pressure was found by phylogenic analysis.Our laboratory will continue to observe antiviral-resistance among circulating influenza viruses.
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Objective To understand the evolutionary characterization of hemagglutinin (HA)gene of pandemic H1N1 influenza virus in Guangdong during 2009-2011. MethodsWe selected 83 pandemic H1N1 strains isolated in Guangdong during 2009-2011. The HA1 genes were sequenced and analyzed comparatively by Bioedit 7.0 and MEGA 4.0. ResultsThe evolutionary rate of Hal gene of pandemic H1N1 and seasonal H1N1 viruses was 5.2×10-3 substitutions/site/year, higher than that of seasonal H1N1 viruses. Most amino acid changes in HA1 molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Two fatal infections were detected with a mutation at HA residue 222, in one virus with a change D222G, and in one virus D222N. ConclusionThe phylogenetic analysis demonstrates that the influenza epidemic in Guangdong at the beginning of 2011 are due to occurrence of genetic changes of pandemic H1N1 virus. The amino acid change at residue 222 of the HA1 are likely to be associated with severe or even fatal illness.
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Objective To analyze the genetic characterization(evolution, antigenicity, enzyme activity sites and glycosylation sites)of the neuraminidase(NA)gene of the novel influenza virus A/H1N1 in 2009 pandemic in Guangdong Province. Methods The viral RNA was extracted from 69 isolates of influenza virus A/H1N1 from patients in 2009 pandemic in Guangdong Province. NA gene fragments were amplified by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. The other 52 NA gene sequences of influenza virus A in different years and different regions were retrieved from GenBank. The analysis of evolution and amino acid sequences were analyzed by MEGA 4.0 software. Results The homology of 2009 novel H1N1 influenza viruses in Guangdong and avian H5N1 influenza virus strains was high(＞85 % ). The amino acid distributions of potential antigenic sites were identical. The enzyme activity sites of NA genes of all virus strains were strictly conserved, which had eight glycosylation sites. But there were amino acid substitutions in 5 glycosylation sites, while it was identical with the 2001 avian H5N1 influenza virus. Conclusion The NA genes of 2009 novel H1N1 influenza viruses in Guangdong are high homologous with avian H5N1 influenza virus and the viral specific binding sites of neuraminidase inhibitor are not changed.
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. Conclusions Two multiplex RT-PCR assays show high consistency with common RT-PCR. The multiplex RT-PCR assays were initially established.