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1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199760

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Haemophilus influenzae/genética , Humanos , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética
2.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: covidwho-1264469

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Haemophilus influenzae/genética , Humanos , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética
3.
J Med Microbiol ; 70(7)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34236300

RESUMO

Introduction. Outbreaks of carbapenem-resistant A. baumannii and A. nosocomialis have occurred worldwide in healthcare settings. Rapid and reliable molecular typing of bacterial isolates is vital for the effective surveillance of institutional outbreaks. The Pan-PCR and OXA-PCR assays are two multiplex PCR-based assays for the molecular typing of Acinetobacter species.Gap statement. However, few studies have investigated the discriminatory power of two multiplex PCR assays in in the genotyping of Acinetobacter species.Aim. We aimed to evaluate the efficacies of the Pan-PCR and OXA-PCR assays for molecular typing of A. baumannii and A. nosocomialis.Methodology. A total of 105 carbapenem-resistant A. baumannii isolates (CRABs) and 93 carbapenem-resistant A. nosocomialis isolates (CRANs) obtained from blood cultures were used for molecular typing by the Pan-PCR and OXA-PCR assays and two multilocus sequence typing (MLST) schemes.Results. The isolates were individually divided into 12 and 21 different sequence types via the Pasteur and Oxford MLST schemes, respectively. Additionally, these isolates were distinguished into 18 different types by the Pan-PCR and OXA-PCR assays. The results of the Pan-PCR and OXA-PCR assays distinguished CRABs and CRANs with a sensitivity of 98.13 % and a specificity of 100 %.Conclusion. The Pan-PCR and OXA-PCR assays are promising alternative methods for rapid molecular typing of CRABs and CRANs in a routine laboratory setting.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter/efeitos dos fármacos , Acinetobacter/genética , Carbapenêmicos/farmacologia , Reação em Cadeia da Polimerase/métodos , Acinetobacter/classificação , Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Humanos , Taiwan/epidemiologia
4.
Medicine (Baltimore) ; 100(25): e26331, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160397

RESUMO

ABSTRACT: Mosaicism can be observed in karyotype analyses of amniotic fluid cells. Differentiating between true mosaicism and pseudomosaicism and determining mosaic proportions can help avoid misjudgments by doctors and effectively reduce mental and physical harm to pregnant women. However, the detection of mosaicism and mosaic proportions via karyotype analysis and fluorescence in situ hybridization (FISH) is extremely complex. We have developed a novel approach, "segmental duplication quantitative fluorescent PCR" (SD-QF-PCR), to detect mosaicism and mosaic proportions.In this study, twenty control samples and fourteen mosaic samples were tested by first-line karyotype analysis; by second-line karyotype analysis, SD-QF-PCR and FISH were used to reassess fetal sex chromosome mosaicism and mosaic proportions.Detection of the 20 control samples by second-line karyotype analysis via FISH and SD-QF-PCR showed normal and consistent results. Among the 14 mosaic samples, the numbers of samples showing true mosaicism and pseudomosaicism detected by the three methods were 6 and 8, respectively.Our study demonstrates that SD-QF-PCR can be used as a complementary method to traditional cytogenetic analysis of amniotic fluid mosaics and has clinical application value.


Assuntos
Cariotipagem/métodos , Mosaicismo , Reação em Cadeia da Polimerase/métodos , Diagnóstico Pré-Natal/métodos , Aberrações dos Cromossomos Sexuais , Amniocentese , Líquido Amniótico/citologia , Células Cultivadas , Estudos de Viabilidade , Feminino , Humanos , Hibridização in Situ Fluorescente , Gravidez , Cultura Primária de Células
5.
PLoS One ; 16(6): e0252757, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34161355

RESUMO

BACKGROUND: A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. METHODS: We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. RESULTS: Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/µL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/µL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. CONCLUSION: Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Calibragem , Proteínas do Envelope de Coronavírus/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
6.
Int J Immunopathol Pharmacol ; 35: 20587384211027679, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34162269

RESUMO

INTRODUCTION: Coronavirus disease 2019 (COVID-19) was declared a global pandemic in March 2020. Since then, several studies have found COVID-19 patients with recurrent viral polymerase chain reaction (PCR) positivity. METHODS: On May 6, 2021, an exhaustive literature search of the Web of Science, PubMed, Cochrane Library, Chinese National Knowledge Infrastructure databases, Embase, Wan Fang Data, VIP database, Sinomed database, BioRxiv, MedRxiv, and Research Square was conducted to find describing the laboratory indicators of recurrent and non-recurrent viral PCR positivity in patients with COVID-19. The data were statistically analyzed using STATA version 15.0. RESULTS: In total, 22 studies-comprising 5154 laboratory-confirmed COVID-19 cases-were included in the analyses. Patients with less severe COVID-19 illness (i.e. those clinically classified as mild or common-type) seemed to exhibit recurrent PCR positivity more commonly than patients with more severe illness (i.e. those classified as severe or critical). There were also significant differences between the two groups in terms of the rates of headaches and dizziness, in addition to the levels of aspartate aminotransferase, C reactive protein, interleukin-6, and lactate dehydrogenase. Further, there were variations in the ratio of CD4+ T cells/CD8+ T cells on admission to the hospital. CONCLUSION: In comparison to COVID-19 patients with non-recurrent viral PCR positivity, patients with recurrent virus PCR positivity seem to experience more severe immune function suppression upon hospital admission.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/imunologia , Imunidade Celular/imunologia , Reação em Cadeia da Polimerase/métodos , COVID-19/epidemiologia , Teste para COVID-19/tendências , Humanos , Reação em Cadeia da Polimerase/tendências , Recidiva
7.
Viruses ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071726

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.


Assuntos
COVID-19/terapia , COVID-19/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/isolamento & purificação , Carga Viral/métodos , Animais , Células Cultivadas , Chlorocebus aethiops , Tecnologia Digital/métodos , Humanos , SARS-CoV-2/genética , Células Vero , Cultura de Vírus
8.
PLoS One ; 16(6): e0252757, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1280620

RESUMO

BACKGROUND: A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. METHODS: We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. RESULTS: Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/µL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/µL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. CONCLUSION: Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Calibragem , Proteínas do Envelope de Coronavírus/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
9.
Biosensors (Basel) ; 11(5)2021 May 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1223947

RESUMO

Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.


Assuntos
Teste de Ácido Nucleico para COVID-19/instrumentação , COVID-19/diagnóstico , Testes Imediatos , Reação em Cadeia da Polimerase/instrumentação , SARS-CoV-2/isolamento & purificação , Animais , Teste de Ácido Nucleico para COVID-19/métodos , Sistemas CRISPR-Cas , Desenho de Equipamento , Humanos , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética
10.
Sci Rep ; 11(1): 12565, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: covidwho-1270671

RESUMO

Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false-negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). We subsequently validated those primers and probes in 211,833 SARS-CoV-2 whole-genome sequences. We obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome from these analyses. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, apicomplexan, and other Betacoronaviruses and less pathogenic sub-strains of coronavirus. The availability of these primer and probe sequences will make it possible to validate more efficient protocols for identifying SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética , Simulação por Computador , Primers do DNA , Genoma Viral , Humanos , SARS-CoV-2/isolamento & purificação , Sequenciamento Completo do Genoma
11.
Viruses ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: covidwho-1256666

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.


Assuntos
COVID-19/terapia , COVID-19/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/isolamento & purificação , Carga Viral/métodos , Animais , Células Cultivadas , Chlorocebus aethiops , Tecnologia Digital/métodos , Humanos , SARS-CoV-2/genética , Células Vero , Cultura de Vírus
12.
Int J Immunopathol Pharmacol ; 35: 20587384211027679, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1286804

RESUMO

INTRODUCTION: Coronavirus disease 2019 (COVID-19) was declared a global pandemic in March 2020. Since then, several studies have found COVID-19 patients with recurrent viral polymerase chain reaction (PCR) positivity. METHODS: On May 6, 2021, an exhaustive literature search of the Web of Science, PubMed, Cochrane Library, Chinese National Knowledge Infrastructure databases, Embase, Wan Fang Data, VIP database, Sinomed database, BioRxiv, MedRxiv, and Research Square was conducted to find describing the laboratory indicators of recurrent and non-recurrent viral PCR positivity in patients with COVID-19. The data were statistically analyzed using STATA version 15.0. RESULTS: In total, 22 studies-comprising 5154 laboratory-confirmed COVID-19 cases-were included in the analyses. Patients with less severe COVID-19 illness (i.e. those clinically classified as mild or common-type) seemed to exhibit recurrent PCR positivity more commonly than patients with more severe illness (i.e. those classified as severe or critical). There were also significant differences between the two groups in terms of the rates of headaches and dizziness, in addition to the levels of aspartate aminotransferase, C reactive protein, interleukin-6, and lactate dehydrogenase. Further, there were variations in the ratio of CD4+ T cells/CD8+ T cells on admission to the hospital. CONCLUSION: In comparison to COVID-19 patients with non-recurrent viral PCR positivity, patients with recurrent virus PCR positivity seem to experience more severe immune function suppression upon hospital admission.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/imunologia , Imunidade Celular/imunologia , Reação em Cadeia da Polimerase/métodos , COVID-19/epidemiologia , Teste para COVID-19/tendências , Humanos , Reação em Cadeia da Polimerase/tendências , Recidiva
13.
Methods Mol Biol ; 2277: 299-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080159

RESUMO

In light of accumulating evidence suggestive of cell type-specific vulnerabilities as a result of normal aging processes that adversely affect the brain, as well as age-related neurodegenerative disorders such as Parkinson's disease (PD), the current chapter highlights how we study mitochondrial DNA (mtDNA) changes at a single-cell level. In particular, we comment on increasing questioning of the narrow neurocentric view of such pathologies, where microglia and astrocytes have traditionally been considered bystanders rather than players in related pathological processes. Here we review the contribution made by single-cell mtDNA alterations towards neuronal vulnerability seen in neurodegenerative disorders, focusing on PD as a prominent example. In addition, we give an overview of methodologies that support such experimental investigations. In considering the significant advances that have been made in recent times for developing mitochondria-specific therapies, investigations to account for cell type-specific mitochondrial patterns and how these are altered by disease hold promise for delivering more effective disease-modifying therapeutics.


Assuntos
Encéfalo/patologia , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Doenças Neurodegenerativas/patologia , Análise de Célula Única/métodos , Envelhecimento/genética , Humanos , Doenças Neurodegenerativas/genética , Doença de Parkinson/genética , Reação em Cadeia da Polimerase/métodos
14.
Methods Mol Biol ; 2277: 433-447, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080167

RESUMO

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Microdissecção e Captura a Laser , Mitocôndrias Musculares/genética , Músculo Esquelético/citologia , Reação em Cadeia da Polimerase/métodos
15.
J Food Sci ; 86(7): 3188-3194, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34146420

RESUMO

Food processors invest significant resources into environmental sampling to detect contamination with potential pathogens, particularly Listeria monocytogenes. To facilitate these efforts, multiple environmental sampling tests (ESTs) have been developed and commercialized that minimize workload, turnaround time, and cost while providing convenient colorimetric detection. For presumptive-positive ESTs, we hypothesized that a relatively minor additional investment could provide, in addition to species confirmation, valuable strain typing data for tracking pathogen spread through a facility, identifying harborage sites, and distinguishing sporadic from persistent or resident contaminants. This hypothesis is based on the demonstrated compatibility of polymorphic locus sequence typing (PLST) with crude samples including food enrichments. Five Listeria ESTs were tested here: broth-based InSite (Hygiena), Path-Chek (Mericon), and Pathfinder (Hardy Diagnositics); and gel-based Petrifilm (3M) and HardyChrom (Hardy Diagnostics). ESTs were inoculated with strains representing two common L. monocytogenes serotypes and nonpathogenic Listeria innocua. Following incubation, broths or suspended colonies were heat treated to inactivate bacteria. Lysates or purified DNAs were prepared and used as templates in PCRs targeting the previously described PLST loci LmiMT1 and LisMT2. Single clear products were obtained from all inoculated ESTs; uninoculated controls were negative. PCR products were subjected to Sanger sequencing, yielding high-quality chromatograms. Phylogenetic analysis confirmed identities to previously determined sequences and revealed relatedness to serotype-matched strains represented in GenBank databases. PRACTICAL APPLICATION: Multiple environmental sampling tests have been commercialized in recent years to facilitate the proactive detection of pathogens, particularly Listeria monocytogenes, within food processing facilities. Coupling a positive detection test with strain typing would enhance its value by providing data that can be used to track pathogen spread through a facility, identify harborage sites, and distinguish sporadic from resident contamination.


Assuntos
Monitoramento Ambiental/métodos , Contaminação de Alimentos/análise , Manipulação de Alimentos/métodos , Listeria/química , Listeria/genética , Tipagem de Sequências Multilocus/métodos , Filogenia , Reação em Cadeia da Polimerase/métodos
16.
Biosensors (Basel) ; 11(5)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069758

RESUMO

A polydimethylsiloxane (PDMS)-based self-priming microfluidic chip with cushion chambers is presented in this study for robust and easy-operation digital polymerase chain reaction (dPCR). The chip has only one inlet and can partition samples autonomously through negative pressure, provided by a de-gassed PDMS layer with a multi-level vertical branching microchannel design. Meanwhile, cushion chambers make the chip capable of very robust use for sample partitioning. Finally, the proposed microfluidic chip showed excellent performance in the absolute quantification of a target gene by performing quantitative detection of a 10-fold serial dilution DNA template. Owing to its characteristics of easy operation, low cost, and high robustness, the proposed dPCR chip is expected to further promote the extensive application of digital PCR, especially in resource-limited settings.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Reação em Cadeia da Polimerase/métodos , Técnicas Biossensoriais , Dimetilpolisiloxanos , Desenho de Equipamento , Microfluídica , Análise de Sequência com Séries de Oligonucleotídeos
17.
Methods Mol Biol ; 2298: 135-151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085243

RESUMO

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (m5C) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of m5C in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.


Assuntos
5-Metilcitosina/metabolismo , Nucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , RNA/genética , Análise de Sequência de DNA/métodos , Biologia Computacional/métodos , Processamento Pós-Transcricional do RNA/genética , Sulfitos/metabolismo
18.
Biosensors (Basel) ; 11(5)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062874

RESUMO

Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.


Assuntos
Teste de Ácido Nucleico para COVID-19/instrumentação , COVID-19/diagnóstico , Testes Imediatos , Reação em Cadeia da Polimerase/instrumentação , SARS-CoV-2/isolamento & purificação , Animais , Teste de Ácido Nucleico para COVID-19/métodos , Sistemas CRISPR-Cas , Desenho de Equipamento , Humanos , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética
19.
Sci Rep ; 11(1): 12565, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131209

RESUMO

Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false-negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). We subsequently validated those primers and probes in 211,833 SARS-CoV-2 whole-genome sequences. We obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome from these analyses. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, apicomplexan, and other Betacoronaviruses and less pathogenic sub-strains of coronavirus. The availability of these primer and probe sequences will make it possible to validate more efficient protocols for identifying SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética , Simulação por Computador , Primers do DNA , Genoma Viral , Humanos , SARS-CoV-2/isolamento & purificação , Sequenciamento Completo do Genoma
20.
Methods Mol Biol ; 2274: 89-100, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050465

RESUMO

Advances in live-cell imaging have been accelerated by the development of various fluorescent indicators. However, indicators that are suitable for multicolor imaging remain a challenge to develop. Herein, we have developed a single fluorescent protein (FP)-based indicator using a semirational molecular design and a molecular evolution approach. We first inserted a ligand-binding domain into the vicinity of an FP chromophore to convert the conformational change induced by ligand binding into a change in fluorescence intensity. We then optimized the linker regions between the FP and the ligand-binding domain to greatly expand the dynamic range (F/F0) of the indicator. Our design and optimization methods are highly versatile and can be used to develop any single FP-based indicators, which will further advance the utility of live-cell imaging.


Assuntos
Corantes Fluorescentes/química , Glucose/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imagem Molecular/métodos , Mutação , Reação em Cadeia da Polimerase/métodos , Evolução Molecular , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida
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