Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 3 de 3
Int J Mol Sci ; 22(11)2021 Jun 04.
Article in English | MEDLINE | ID: covidwho-1264469


Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3' Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.

DNA-Directed DNA Polymerase/metabolism , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Alleles , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Bacterial/metabolism , Haemophilus influenzae/genetics , Humans , Meningitis, Bacterial/diagnosis , Meningitis, Bacterial/microbiology , Neisseria meningitidis/genetics , Streptococcus pneumoniae/genetics
Biosens Bioelectron ; 178: 113001, 2021 Apr 15.
Article in English | MEDLINE | ID: covidwho-1064880


Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.

COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , CRISPR-Cas Systems , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , COVID-19 Nucleic Acid Testing/instrumentation , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Food Microbiology , Humans , Magnetics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Polypropylenes , RNA, Viral/genetics , RNA, Viral/isolation & purification , Salmonella typhimurium/genetics , Salmonella typhimurium/isolation & purification , Self-Testing
Gut ; 70(4): 698-706, 2021 04.
Article in English | MEDLINE | ID: covidwho-1024254


OBJECTIVE: Although COVID-19 is primarily a respiratory illness, there is mounting evidence suggesting that the GI tract is involved in this disease. We investigated whether the gut microbiome is linked to disease severity in patients with COVID-19, and whether perturbations in microbiome composition, if any, resolve with clearance of the SARS-CoV-2 virus. METHODS: In this two-hospital cohort study, we obtained blood, stool and patient records from 100 patients with laboratory-confirmed SARS-CoV-2 infection. Serial stool samples were collected from 27 of the 100 patients up to 30 days after clearance of SARS-CoV-2. Gut microbiome compositions were characterised by shotgun sequencing total DNA extracted from stools. Concentrations of inflammatory cytokines and blood markers were measured from plasma. RESULTS: Gut microbiome composition was significantly altered in patients with COVID-19 compared with non-COVID-19 individuals irrespective of whether patients had received medication (p<0.01). Several gut commensals with known immunomodulatory potential such as Faecalibacterium prausnitzii, Eubacterium rectale and bifidobacteria were underrepresented in patients and remained low in samples collected up to 30 days after disease resolution. Moreover, this perturbed composition exhibited stratification with disease severity concordant with elevated concentrations of inflammatory cytokines and blood markers such as C reactive protein, lactate dehydrogenase, aspartate aminotransferase and gamma-glutamyl transferase. CONCLUSION: Associations between gut microbiota composition, levels of cytokines and inflammatory markers in patients with COVID-19 suggest that the gut microbiome is involved in the magnitude of COVID-19 severity possibly via modulating host immune responses. Furthermore, the gut microbiota dysbiosis after disease resolution could contribute to persistent symptoms, highlighting a need to understand how gut microorganisms are involved in inflammation and COVID-19.

Bacteria , COVID-19 , Dysbiosis , Gastrointestinal Microbiome/immunology , Gastrointestinal Tract , Immunity , SARS-CoV-2 , Adult , Bacteria/genetics , Bacteria/immunology , Bacteria/isolation & purification , C-Reactive Protein/analysis , COVID-19/blood , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/immunology , Cytokines/analysis , DNA, Bacterial/isolation & purification , Dysbiosis/epidemiology , Dysbiosis/etiology , Dysbiosis/immunology , Dysbiosis/virology , Female , Gastrointestinal Tract/immunology , Gastrointestinal Tract/microbiology , Gastrointestinal Tract/virology , Hong Kong , Humans , Male , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Severity of Illness Index , Transferases/analysis