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1.
PLoS One ; 16(9): e0249143, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34473726

RESUMEN

Pork processing plants were apparent hotspots for SARS-CoV2 in the spring of 2020. As a result, the swine industry was confronted with a major occupational health, financial, and animal welfare crisis. The objective of this work was to describe the epidemiological situation within processing plants, develop mathematical models to simulate transmission in these plants, and test the effectiveness of routine PCR screening at minimizing SARS-CoV2 circulation. Cumulative incidence of clinical (PCR-confirmed) disease plateaued at ~2.5% to 25% across the three plants studied here. For larger outbreaks, antibody prevalence was approximately 30% to 40%. Secondly, we developed a mathematical model that accounts for asymptomatic, pre-symptomatic, and background "community" transmission. By calibrating this model to observed epidemiological data, we estimated the initial reproduction number (R) of the virus. Across plants, R generally ranged between 2 and 4 during the initial phase, but subsequently declined to ~1 after two to three weeks, most likely as a result of implementation/compliance with biosecurity measures in combination with population immunity. Using the calibrated model to simulate a range of possible scenarios, we show that the effectiveness of routine PCR-screening at minimizing disease spread was far more influenced by testing frequency than by delays in results, R, or background community transmission rates. Testing every three days generally averted about 25% to 40% of clinical cases across a range of assumptions, while testing every 14 days typically averted 7 to 13% of clinical cases. However, the absolute number of additional clinical cases expected and averted was influenced by whether there was residual immunity from a previous peak (i.e., routine testing is implemented after the workforce had experienced an initial outbreak). In contrast, when using PCR-screening to prevent outbreaks or in the early stages of an outbreak, even frequent testing may not prevent a large outbreak within the workforce. This research helps to identify protocols that minimize risk to occupational safety and health and support continuity of business for U.S. processing plants. While the model was calibrated to meat processing plants, the structure of the model and insights about testing are generalizable to other settings where large number of people work in close proximity.


Asunto(s)
Algoritmos , COVID-19/diagnóstico , Industria de Procesamiento de Alimentos , Tamizaje Masivo/métodos , Modelos Teóricos , Salud Laboral/estadística & datos numéricos , Carne de Cerdo , Animales , Anticuerpos Antivirales/inmunología , COVID-19/transmisión , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19/métodos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , Sensibilidad y Especificidad , Porcinos
2.
Methods Mol Biol ; 2327: 119-137, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34410643

RESUMEN

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.


Asunto(s)
Genoma Viral , SARS-CoV-2/genética , Saliva/virología , Secuenciación Completa del Genoma/métodos , Biología Computacional , ADN Complementario/genética , Humanos , Sondas Moleculares/genética , Reacción en Cadena de la Polimerasa/métodos , SARS-CoV-2/aislamiento & purificación , Manejo de Especímenes/métodos , Estreptavidina , Secuenciación Completa del Genoma/instrumentación
3.
Cochrane Database Syst Rev ; 8: CD013207, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34383961

RESUMEN

BACKGROUND: The standard method of diagnosing HIV in infants and children less than 18 months is with a nucleic acid amplification test reverse transcriptase polymerase chain reaction test (NAT RT-PCR) detecting viral ribonucleic acid (RNA). Laboratory testing using the RT-PCR platform for HIV infection is limited by poor access, logistical support, and delays in relaying test results and initiating therapy in low-resource settings. The use of rapid diagnostic tests at or near the point-of-care (POC) can increase access to early diagnosis of HIV infection in infants and children less than 18 months of age and timely initiation of antiretroviral therapy (ART). OBJECTIVES: To summarize the diagnostic accuracy of point-of-care nucleic acid-based testing (POC NAT) to detect HIV-1/HIV-2 infection in infants and children aged 18 months or less exposed to HIV infection. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (until 2 February 2021), MEDLINE and Embase (until 1 February 2021), and LILACS and Web of Science (until 2 February 2021) with no language or publication status restriction. We also searched conference websites and clinical trial registries, tracked reference lists of included studies and relevant systematic reviews, and consulted experts for potentially eligible studies. SELECTION CRITERIA: We defined POC tests as rapid diagnostic tests conducted at or near the patient site. We included any primary study that compared the results of a POC NAT to a reference standard of laboratory NAT RT-PCR or total nucleic acid testing to detect the presence or absence of HIV infection denoted by HIV viral nucleic acids in infants and children aged 18 months or less who were exposed to HIV-1/HIV-2 infection. We included cross-sectional, prospective, and retrospective study designs and those that provided sufficient data to create the 2 × 2 table to calculate sensitivity and specificity. We excluded diagnostic case control studies with healthy controls. DATA COLLECTION AND ANALYSIS: We extracted information on study characteristics using a pretested standardized data extraction form. We used the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) tool to assess the risk of bias and applicability concerns of the included studies. Two review authors independently selected and assessed the included studies, resolving any disagreements by consensus. The unit of analysis was the participant. We first conducted preliminary exploratory analyses by plotting estimates of sensitivity and specificity from each study on forest plots and in receiver operating characteristic (ROC) space. For the overall meta-analyses, we pooled estimates of sensitivity and specificity using the bivariate meta-analysis model at a common threshold (presence or absence of infection). MAIN RESULTS: We identified a total of 12 studies (15 evaluations, 15,120 participants). All studies were conducted in sub-Saharan Africa. The ages of included infants and children in the evaluations were as follows: at birth (n = 6), ≤ 12 months (n = 3), ≤ 18 months (n = 5), and ≤ 24 months (n = 1). Ten evaluations were field evaluations of the POC NAT test at the point of care, and five were laboratory evaluations of the POC NAT tests.The POC NAT tests evaluated included Alere q HIV-1/2 Detect qualitative test (recently renamed m-PIMA q HIV-1/2 Detect qualitative test) (n = 6), Xpert HIV-1 qualitative test (n = 6), and SAMBA HIV-1 qualitative test (n = 3). POC NAT pooled sensitivity and specificity (95% confidence interval (CI)) against laboratory reference standard tests were 98.6% (96.1 to 99.5) (15 evaluations, 1728 participants) and 99.9% (99.7 to 99.9) (15 evaluations, 13,392 participants) in infants and children ≤ 18 months. Risk of bias in the included studies was mostly low or unclear due to poor reporting. Five evaluations had some concerns for applicability for the index test, as they were POC tests evaluated in a laboratory setting, but there was no difference detected between settings in sensitivity (-1.3% (95% CI -4.1 to 1.5)); and specificity results were similar. AUTHORS' CONCLUSIONS: For the diagnosis of HIV-1/HIV-2 infection, we found the sensitivity and specificity of POC NAT tests to be high in infants and children aged 18 months or less who were exposed to HIV infection.


Asunto(s)
Infecciones por VIH/diagnóstico , VIH-1/genética , VIH-2/genética , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa/métodos , Estudios Transversales , Femenino , VIH-1/aislamiento & purificación , VIH-2/aislamiento & purificación , Humanos , Lactante , Recién Nacido , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad
4.
Microbiol Spectr ; 9(1): e0016221, 2021 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-34406838

RESUMEN

The continued need for molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the potential for self-collected saliva as an alternative to nasopharyngeal (NP) swabs for sample acquisition led us to compare saliva to NP swabs in an outpatient setting without restrictions to avoid food, drink, smoking, or tooth-brushing. A total of 385 pairs of NP and saliva specimens were obtained, the majority from individuals presenting for initial evaluation, and were tested on two high-sensitivity reverse transcriptase PCR (RT-PCR) platforms, the Abbott m2000 and Abbott Alinity m (both with limits of detection [LoD] of 100 copies of viral RNA/ml). Concordance between saliva and NP swabs was excellent overall (Cohen's κ = 0.93) for both initial and follow-up testing, for both platforms, and for specimens treated with guanidinium transport medium as preservative as well as for untreated saliva (κ = 0.88 to 0.95). Viral loads were on average 16× higher in NP specimens than saliva specimens, suggesting that only the relatively small fraction of outpatients (∼8% in this study) who present with very low viral loads (<1,600 copies/ml from NP swabs) would be missed by testing saliva instead of NP swabs when using sensitive testing platforms. Special attention was necessary to ensure leak-resistant specimen collection and transport. The advantages of self-collection of saliva, without behavioral restrictions, will likely outweigh a minor potential decrease in clinical sensitivity in individuals less likely to pose an infectious risk to others for many real-world scenarios, especially for initial testing. IMPORTANCE In general, the most accurate COVID-19 testing is hands-on and uncomfortable, requiring trained staff and a "brain-tickling" nasopharyngeal swab. Saliva would be much easier on both fronts, since patients could collect it themselves, and it is after all just spit. However, despite much interest, it remains unclear how well saliva performs in real-world settings when just using it in place of an NP swab without elaborate or cumbersome restrictions about not eating/drinking before testing, etc. Also, almost all studies of COVID-19 testing, whether of NP swabs, saliva, or otherwise, have been restricted to reporting results in the abstruse units of "CT values," which only mean something in the context of a specific assay and testing platform. Here, we compared saliva versus NP swabs in a real-world setting without restriction and report all results in natural units-the amount of virus being shed-showing that saliva is essentially just as good as NP swabs.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Saliva/virología , Manejo de Especímenes/métodos , Pruebas Diagnósticas de Rutina , Humanos , Límite de Detección , Reacción en Cadena de la Polimerasa/métodos , ARN Viral , Sensibilidad y Especificidad , Tiempo , Carga Viral
5.
Microbiol Spectr ; 9(1): e0006221, 2021 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-34431689

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic has challenged clinical diagnostic operations due to supply shortages and high staffing needs to collect nasopharyngeal (NP) swab samples. Saliva is an easily accessible alternative specimen type to overcome some of these challenges. In this study, we first used paired saliva and NP swab specimens (n = 128) to compare test performance characteristics with three RNA extraction platforms, i.e., Maxwell RSC (Promega), MagNA Pure 96 (Roche), and KingFisher Flex (Thermo Fisher Scientific), together with two PCR chemistries, i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2019-nCoV) Centers for Disease Control and Prevention (CDC) quantitative PCR (qPCR) probe assay (Integrated DNA Technologies) and TagPath COVID-19 combination kit (Thermo Fisher Scientific). This study demonstrated that both saliva and NP swab specimens performed well, with 97% agreement when tested by the CDC qPCR chemistry using Maxwell and MagNA Pure RNA extraction platforms. We then compared 12 weeks of saliva and NP swab testing results using two independent asymptomatic populations, including a community surveillance program using saliva samples only (n = 466) and preoperative screening using NP swab samples only (n = 8,461). The positive detection rates among participants with either saliva or NP swab samples were 1.07% and 1.12%, respectively, which confirms the low pretest probabilities for COVID-19 infections in asymptomatic populations. Notably, there was no increased proportion of low-titer cases (inconclusive results) reported in the asymptomatic groups, compared with the all-comers groups (0.21% and 0.66%, respectively, in the community population and 0.25% and 0.49%, respectively, in the preoperative population); this suggests that low-viral-titer carriers can be found similarly in both groups with saliva or NP swab specimens. In summary, saliva can be considered a good alternative for noninvasive but well-instructed self-collection. IMPORTANCE Our study shows that saliva is a noninvasive respiratory secretion sample type that contains equal or more host materials (RNase P), compared with those contained in the corresponding NP swab specimens, in 103 paired samples. SARS-CoV-2 detection with two RNA extraction platforms, Maxwell and MagNA Pure, with CDC qPCR chemistry showed similar test sensitivities for paired specimens. We then analyzed SARS-CoV-2 detections rates in two independent groups of asymptomatic participants, i.e., a group at a community screening station with supervised saliva collection only (n = 466) and a preoperative screening group (n = 8,461) with NP swabbing only. Similar detection rates of 1.07% for the community group and 1.12% for the preoperative group supported the similar test performances in these groups predicted to have low pretest probabilities of infection. With mindful preparation, saliva can be considered for schools and clinical participants when adequate collection education can be provided and compliance can be established.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virología , Reacción en Cadena de la Polimerasa/métodos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Saliva/virología , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Masculino , Tamizaje Masivo , Persona de Mediana Edad , Pandemias , ARN Viral/análisis , Manejo de Especímenes/métodos , Adulto Joven
7.
Viruses ; 13(7)2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34372583

RESUMEN

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.


Asunto(s)
Braquiuros/virología , Coinfección/epidemiología , Penaeidae/virología , Animales , Animales Salvajes/virología , Acuicultura/métodos , Densovirinae/genética , Densovirinae/patogenicidad , India , Reacción en Cadena de la Polimerasa/métodos , Virus del Síndrome de la Mancha Blanca 1/genética , Virus del Síndrome de la Mancha Blanca 1/patogenicidad
8.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-34199760

RESUMEN

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.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Alelos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Haemophilus influenzae/genética , Humanos , Meningitis Bacterianas/diagnóstico , Meningitis Bacterianas/microbiología , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética
10.
Genes (Basel) ; 12(6)2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-34204948

RESUMEN

Species identification of the components of various duck breeds has revealed that the lowest identifiable number of components depends on the breed. The results (shown on the agarose gel) of a species-specific PCR reaction for Rouen ducks were less intense than the results for the same amount of components from other popular duck breeds, suggesting differences in the Rouen duck genome. Therefore, the present study aimed to identify part of the Rouen duck's gene sequences and to develop two new primer pairs. The first pair enables breed-independent identification of duck DNA, and the second distinguishes Rouen ducks from Chinese and Indian Runner ducks. The sequencing reaction yielded sequences of 1386 bp in length, and the identified sequence differs by around 7% from the sequences of Chinese duck species. The detected sequence contributes to improving species identification methods for duck DNA. On its basis, two primers for the identification of duck DNA were designed. The first allows for DNA amplification with the same sensitivity regardless of duck breed. The second primer's pair is breed specific, and it distinguishes Rouen ducks from Chinese and Indian Runner ducks. Both methods are very sensitive (0.05%).


Asunto(s)
Cruzamiento/métodos , ADN Mitocondrial/genética , Patos/genética , Técnicas de Genotipaje/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Técnicas de Genotipaje/normas , Estándares de Referencia
11.
Nat Biomed Eng ; 5(7): 702-712, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34211146

RESUMEN

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics.


Asunto(s)
ADN/análisis , Reacción en Cadena de la Polimerasa/métodos , Bacterias/genética , Bacterias/aislamiento & purificación , ADN/metabolismo , Sondas de ADN/metabolismo , Colorantes Fluorescentes/química , Genoma Humano , Genotipo , Humanos , Límite de Detección , Análisis por Micromatrices , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa/instrumentación , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados
12.
J Med Microbiol ; 70(7)2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34236300

RESUMEN

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.


Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Carbapenémicos/farmacología , Reacción en Cadena de la Polimerasa/métodos , Acinetobacter/clasificación , Infecciones por Acinetobacter/epidemiología , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/clasificación , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Humanos , Taiwán/epidemiología
13.
Biomed Res Int ; 2021: 5568350, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34327228

RESUMEN

In this COVID-19 pandemic, there is a dire need for cost-effective and less time-consuming alternatives for SARS-CoV-2 testing. The RNA extraction-free method for detecting SARS-CoV-2 in saliva is a promising option. This study found that it has high sensitivity (85.34%), specificity (95.04%), and was comparable to the gold standard nasopharyngeal swab (NPS) sample tests. The method showed good agreement between salivary and NPS samples, with a kappa coefficient of 0.797. However, there are variations in the sensitivity and specificity based on the RT-PCR kit used. The Thermo Fisher Applied Biosystems showed high sensitivity, positive predictive value (PPV), and negative predictive value (NPV) but also showed a higher percentage of invalid reports. On the other hand, the BGI kit showed high specificity, better agreement (kappa coefficient) between the results of saliva and NPS samples, and higher correlation between the Ct values of saliva and NPS samples. Thus, the RNA extraction-free method for salivary sample serves as an effective alternative screening method for COVID-19.


Asunto(s)
Prueba de COVID-19/métodos , COVID-19/virología , ARN Viral/aislamiento & purificación , SARS-CoV-2/aislamiento & purificación , Saliva/virología , COVID-19/diagnóstico , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19 , Análisis Costo-Beneficio , Pruebas Diagnósticas de Rutina , Humanos , Tamizaje Masivo/métodos , Nasofaringe/virología , Pandemias , Reacción en Cadena de la Polimerasa/métodos , Valor Predictivo de las Pruebas , ARN Viral/genética , Juego de Reactivos para Diagnóstico , SARS-CoV-2/genética , Sensibilidad y Especificidad , Manejo de Especímenes/métodos
14.
Methods Mol Biol ; 2277: 299-329, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34080159

RESUMEN

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.


Asunto(s)
Encéfalo/patología , ADN Mitocondrial/análisis , ADN Mitocondrial/genética , Enfermedades Neurodegenerativas/patología , Análisis de la Célula Individual/métodos , Envejecimiento/genética , Humanos , Enfermedades Neurodegenerativas/genética , Enfermedad de Parkinson/genética , Reacción en Cadena de la Polimerasa/métodos
15.
Methods Mol Biol ; 2277: 433-447, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34080167

RESUMEN

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 .


Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Captura por Microdisección con Láser , Mitocondrias Musculares/genética , Músculo Esquelético/citología , Reacción en Cadena de la Polimerasa/métodos
16.
Medicine (Baltimore) ; 100(25): e26331, 2021 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-34160397

RESUMEN

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.


Asunto(s)
Cariotipificación/métodos , Mosaicismo , Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Prenatal/métodos , Aberraciones Cromosómicas Sexuales , Amniocentesis , Líquido Amniótico/citología , Células Cultivadas , Estudios de Factibilidad , Femenino , Humanos , Hibridación Fluorescente in Situ , Embarazo , Cultivo Primario de Células
17.
Int J Biol Macromol ; 184: 750-759, 2021 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-34171259

RESUMEN

Pathogens pose a severe threat to food safety and human health. The traditional methods for pathogen detection can't meet the growing diagnosis and control need. Digital PCR (dPCR) attracts a considerable attention for its ability to absolutely quantify pathogens with features of high selectivity, simplicity, accuracy and rapidity. The dPCR technique that achieves absolute quantification based on end-point measurement without standard curve offers a guideline for further genetic analysis and molecular diagnosis. It could contribute to the quantification of low level of nucleic acid, early detection and timely prevention of pathogenic diseases. In this review, 1442 publications about dPCR were selected and the detections of various pathogens by dPCR were reviewed comprehensively, including viruses, bacteria, parasites and fungi. A number of examples are cited to illustrate that dPCR is a new powerful tool with desired accuracy, sensitivity, and reproducibility for quantification of different types of pathogens. Moreover, the benefits, challenges and future prospects of the dPCR were also highlighted in this review.


Asunto(s)
Infecciones/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Bacterias/aislamiento & purificación , Microbiología de Alimentos , Parasitología de Alimentos , Hongos/aislamiento & purificación , Humanos , Parásitos/aislamiento & purificación , Reproducibilidad de los Resultados , Virus/aislamiento & purificación
18.
Genes (Basel) ; 12(5)2021 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-34065649

RESUMEN

The aroma of grapes and derived wines has long been one of the major traits considered in the selection of grapevine varieties through the centuries. In particular, Muscat aromatic grapes have been highly appreciated and widespread since ancient times. Monoterpenes are the key compounds responsible for the Muscat flavor. A major QTL affecting monoterpene level has been found to co-localize with the 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) gene, encoding for the 1-deoxy-D-xylulose 5-phosphate synthase enzyme involved in the plastidial pathway of terpene biosynthesis. In more detail, a single nucleotide polymorphism (SNP 1822) in the coding region of the gene causes a "gain of function" mutation, which is involved in Muscat flavor. In this work, we have developed a digital PCR-based assay to target allelic variations in the VvDXS gene, SNP1822, with the aim to propose a fast and sensitive analytical tool for targeting Muscat-flavored grapevine genotypes. The assay accurately predicts the genetic structure at 1822 SNP, critical for the development of the aroma in the great majority of Muscats. In the case of grapes in which the aromatic component is due to mutations other than SNP 1822 (e.g., Chasselas Musqué and Chardonnay Muscat), further specific assays can be developed.


Asunto(s)
Técnicas de Genotipaje/métodos , Proteínas de Plantas/genética , Polimorfismo de Nucleótido Simple , Transferasas/genética , Vitis/genética , Mutación con Ganancia de Función , Fitomejoramiento/métodos , Reacción en Cadena de la Polimerasa/métodos , Vitis/metabolismo
19.
Diabetes ; 70(6): 1357-1371, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34162681

RESUMEN

Diabetic foot infections (DFIs) cause substantial morbidity and mortality. The mainstay of the treatment is empiric antibiotics and surgical debridement in severe cases. In this study, we performed nanopore 16S rDNA sequencing from the debridement specimens of DFIs. Fifty-four surgical debridement specimens obtained from 45 patients with medically intractable DFI were included. The 16S rDNA PCR was performed on each specimen, and Nanopore sequencing was performed for up to 3 h. The reads were aligned to the BLAST database, and the results were compared with conventional culture studies. The 16S sequencing results revealed that the majority of the DFIs (44 of 54, 81.5%) were polymicrobial infections. All bacteria isolated by conventional culture studies were detected by 16S sequencing. Several anaerobes (Prevotella, Finegoldia, Anaerococcus, Bacteroides) were commonly identified by 16S sequencing but were frequently missed by culture studies. In many cases, certain bacteria only revealed by the 16S sequencing were more abundant than the bacteria isolated by the culture studies. In conclusion, nanopore 16S sequencing was capable of pathogen identification in DFIs and has many advantages over conventional culture studies. Nanopore 16S sequencing enables a comprehensive understanding of the bacteria involved in DFIs.


Asunto(s)
Bacterias/genética , Pie Diabético/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Adulto , Anciano , Anciano de 80 o más Años , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Estudios de Cohortes , Tratamiento Conservador , Pie Diabético/patología , Pie Diabético/terapia , Femenino , Humanos , Masculino , Técnicas Microbiológicas/métodos , Microbiota/genética , Persona de Mediana Edad , Secuenciación de Nanoporos/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , Insuficiencia del Tratamiento
20.
PLoS One ; 16(6): e0252757, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34161355

RESUMEN

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.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Calibración , Proteínas de la Envoltura de Coronavirus/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad
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