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1.
Sci Rep ; 11(1): 13884, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230585

RESUMO

This is the first report of SARS-CoV-2 detection on field-collected Musca domestica housefly surface and tissue samples using the high-sensitive PCR assay which suggests the possible insect-borne transmission. The study was conducted in Shiraz city, southern Iran, in May and Jun 2020. Adult flies were sampled at the outdoor areas of two hospitals treating COVID-19 patients. Fly samples were first washed twice to remove the insect surface attached to SARS-CoV-2 virions. After that, the disinfected fly samples were homogenized. Fly surface washout and homogenate samples were tested using Taq Man real-time PCR assay for the SARS-CoV-2 virus. In a total of 156 houseflies, 75% of samples from the body washout samples were positive for SARS-CoV-2. Strikingly, 37% of the homogenized specimens were positive for the SARS-CoV-2, suggesting the possible infection of the insects or uptake of the virion to the insect metabolism. The other possibility is the houseflies up took the blood or blood fluids of the patients and the RNA of the SARS-CoV-2 survived in the insect body without replicating. Our preliminary findings suggest that the houseflies could transmit SARS-CoV-2 as a mechanical or biological vector especially during the warm seasons while increasing the population and activity of houseflies.


Assuntos
Moscas Domésticas/virologia , Insetos Vetores/virologia , Técnicas de Diagnóstico Molecular , SARS-CoV-2/patogenicidade , Animais , Humanos , Irã (Geográfico) , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estações do Ano
2.
Cochrane Database Syst Rev ; 5: CD012972, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-34097769

RESUMO

BACKGROUND: The World Health Organization (WHO) recommends Xpert MTB/RIF in place of smear microscopy to diagnose tuberculosis (TB), and many countries have adopted it into their diagnostic algorithms. However, it is not clear whether the greater accuracy of the test translates into improved health outcomes. OBJECTIVES: To assess the impact of Xpert MTB/RIF on patient outcomes in people being investigated for tuberculosis. SEARCH METHODS: We searched the following databases, without language restriction, from 2007 to 24 July 2020: Cochrane Infectious Disease Group (CIDG) Specialized Register; CENTRAL; MEDLINE OVID; Embase OVID; CINAHL EBSCO; LILACS BIREME; Science Citation Index Expanded (Web of Science), Social Sciences citation index (Web of Science), and Conference Proceedings Citation Index - Social Science & Humanities (Web of Science). We also searched the WHO International Clinical Trials Registry Platform, ClinicalTrials.gov, and the Pan African Clinical Trials Registry for ongoing trials. SELECTION CRITERIA: We included individual- and cluster-randomized trials, and before-after studies, in participants being investigated for tuberculosis. We analysed the randomized and non-randomized studies separately.  DATA COLLECTION AND ANALYSIS: For each study, two review authors independently extracted data, using a piloted data extraction tool. We assessed the risk of bias using Cochrane and Effective Practice and Organisation of Care (EPOC) tools. We used random effects meta-analysis to allow for heterogeneity between studies in setting and design.  The certainty of the  evidence in the randomized trials was assessed by GRADE. MAIN RESULTS: We included 12 studies: eight were randomized controlled trials (RCTs), and four were before-and-after studies. Most included RCTs had a low risk of bias in most domains of the Cochrane 'Risk of bias' tool. There was inconclusive evidence of an effect of Xpert MTB/RIF on all-cause mortality, both overall (risk ratio (RR) 0.89, 95% confidence interval (CI) 0.75 to 1.05; 5 RCTs, 9932 participants, moderate-certainty evidence), and restricted to studies with six-month follow-up (RR 0.98, 95% CI 0.78 to 1.22; 3 RCTs, 8143 participants; moderate-certainty evidence). There was probably a reduction in mortality in participants known to be infected with HIV (odds ratio (OR) 0.80, 95% CI 0.67 to 0.96; 5 RCTs, 5855 participants; moderate-certainty evidence). It is uncertain whether Xpert MTB/RIF has no or a modest effect on the proportion of participants starting tuberculosis treatment who had a successful treatment outcome (OR) 1.10, 95% CI 0.96 to 1.26; 3RCTs, 4802 participants; moderate-certainty evidence). There was also inconclusive evidence of an effect on the  proportion of participants who were treated for tuberculosis (RR 1.10, 95% CI 0.98 to 1.23; 5 RCTs, 8793 participants; moderate-certainty evidence). The proportion of participants treated for tuberculosis who had bacteriological confirmation was probably higher in the Xpert MTB/RIF group (RR 1.44, 95% CI 1.29 to 1.61; 6 RCTs, 2068 participants; moderate-certainty evidence). The proportion of participants with bacteriological confirmation who were lost to follow-up pre-treatment was probably reduced (RR 0.59, 95% CI 0.41 to 0.85; 3 RCTs, 1217 participants; moderate-certainty evidence). AUTHORS' CONCLUSIONS: We were unable to confidently rule in or rule out the effect on all-cause mortality of using Xpert MTB/RIF rather than smear microscopy. Xpert MTB/RIF probably reduces mortality among participants known to be infected with HIV. We are uncertain whether Xpert MTB/RIF has a modest effect or not on the proportion treated or, among those treated, on the proportion with a successful outcome. It probably does not have a substantial effect on these outcomes. Xpert MTB/RIF probably increases both the proportion of treated participants who had bacteriological confirmation, and the proportion with a laboratory-confirmed diagnosis who were treated. These findings may inform decisions about uptake alongside evidence on cost-effectiveness and implementation.


Assuntos
Antibióticos Antituberculose/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rifampina/farmacologia , Tuberculose Pulmonar/diagnóstico , Viés , Intervalos de Confiança , Estudos Controlados Antes e Depois , Farmacorresistência Bacteriana , Infecções por HIV/mortalidade , Humanos , Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Razão de Chances , Ensaios Clínicos Controlados Aleatórios como Assunto , Kit de Reagentes para Diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/mortalidade
3.
Sci Rep ; 11(1): 11899, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099796

RESUMO

The pandemic caused by SARS-CoV-2 resulted in increasing demands for diagnostic tests, leading to a shortage of recommended testing materials and reagents. This study reports on the performance of self-sampled alternative swabbing material (ordinary Q-tips tested against flocked swab and rayon swab), of reagents for classical RNA extraction (phenol/guanidine-based protocol against a commercial kit), and of intercalating dye-based one-step quantitative reverse transcription real-time PCRs (RT-qPCR) compared against the gold standard hydrolysis probe-based assays for SARS-CoV-2 detection. The study found sampling with Q-tips, RNA extraction with classical protocol and intercalating dye-based RT-qPCR as a reliable and comparably sensitive strategy for detection of SARS-CoV-2-particularly valuable in the current period with a resurgent and dramatic increase in SARS-CoV-2 infections and growing shortage of diagnostic materials especially for regions limited in resources.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/patogenicidade , Manejo de Espécimes , Teste para COVID-19/métodos , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/fisiologia , Manejo de Espécimes/métodos , Fatores de Tempo
4.
Sci Rep ; 11(1): 12676, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135391

RESUMO

Regular PCR testing of nasopharyngeal swabs from symptomatic individuals for SARS-CoV-2 virus has become the established method by which health services are managing the COVID-19 pandemic. Businesses such as AstraZeneca have also prioritised voluntary asymptomatic testing to keep workplaces safe and maintain supply of essential medicines to patients. We describe the development of an internal automated SARS-CoV-2 testing programme including the transformative introduction of saliva as an alternative sample type.


Assuntos
Doenças Assintomáticas/epidemiologia , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/epidemiologia , Pandemias/prevenção & controle , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Saliva/virologia , Recursos Humanos , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , Nasofaringe/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos
5.
Methods Mol Biol ; 2268: 21-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085259

RESUMO

A workflow is described for assaying the expression of G protein-coupled receptors (GPCRs) in cultured cells, using a combination of methods that assess GPCR mRNAs. Beginning from the isolation of cDNA and preparation of mRNA, we provide protocols for designing and testing qPCR primers, assaying mRNA expression using qPCR and high-throughput analysis of GPCR mRNA expression via TaqMan qPCR-based, GPCR-selective arrays. We also provide a workflow for analysis of expression from RNA-sequencing (RNA-seq) assays, which can be queried to yield expression of GPCRs and related genes in samples of interest, as well as to test changes in expression between groups, such as in cells treated with drugs or from healthy and diseased subjects. We place priority on optimized protocols that distinguish signal from noise, as GPCR mRNAs are typically present in low abundance, necessitating techniques that maximize sensitivity while minimizing noise. These methods may also be applicable for assessing the expression of members of families of other low abundance genes via high-throughput analyses of mRNAs, followed by independent confirmation and validation of results via qPCR.


Assuntos
Análise em Microsséries/métodos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Sequência de RNA/métodos , Humanos , Cultura Primária de Células , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/genética
6.
Biomed Res Int ; 2021: 6653950, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34124257

RESUMO

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Assuntos
COVID-19/diagnóstico , Proteínas do Envelope de Coronavírus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , 2-Propanol/química , Biomarcadores/análise , COVID-19/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Humanos , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Viruses ; 13(5)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062916

RESUMO

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96-98), and a sensitivity of 85% (95% CI: 82-89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86-95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657-41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , Idoso , Antígenos Virais/análise , COVID-19/genética , COVID-19/imunologia , Serviço Hospitalar de Emergência , Feminino , Humanos , Itália/epidemiologia , Masculino , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade
8.
Viruses ; 13(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067983

RESUMO

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/sangue , COVID-19/genética , Ensaios de Triagem em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , RNA Viral/sangue , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
9.
Toxins (Basel) ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071223

RESUMO

Resistance against infection by the fungus Aspergillus flavus Link in commercial maize (Zea mays L.) is the topic of many studies, but few studies have investigated the effects of A. flavus infection on gene expression levels in ear kernels. A crucial component of gene expression profiling by RT-qPCR is having a reliable set of reference genes that show relatively constant expression across the treatments and phenotypes under study. Currently, however, there is no published information on reference genes suitable for measuring changes in kernel gene expression levels after infection with A. flavus. Thus, in this study, six candidate reference genes (ACT1, ß-Tub2, eIF4A2, TATA, EFIα, and GAPDH) were evaluated and ranked according to their expression stability. The genes were amplified from first-strand cDNA samples synthesized from kernels of two susceptible and two resistant maize lines that were either inoculated with A. flavus or water or not inoculated. Three software packages were used to calculate and rank the stability of expression for these genesgeNorm, NormFinder, and BestKeeper. The analysis revealed that the most stable genes to normalize expression levels from maize kernels responding to A. flavus inoculation and wounding were ACT1, EFIα, and eIF4A2.


Assuntos
Aspergillus flavus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zea mays/genética , Zea mays/microbiologia , Perfilação da Expressão Gênica
10.
Viruses ; 13(5)2021 05 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1224249

RESUMO

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96-98), and a sensitivity of 85% (95% CI: 82-89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86-95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657-41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , Idoso , Antígenos Virais/análise , COVID-19/genética , COVID-19/imunologia , Serviço Hospitalar de Emergência , Feminino , Humanos , Itália/epidemiologia , Masculino , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade
11.
Viruses ; 13(5)2021 05 19.
Artigo em Inglês | MEDLINE | ID: covidwho-1234837

RESUMO

SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a "good concordance" (Cohen's k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen's k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen's k for this was "very good concordance". The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Humanos , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Carga Viral , Proteínas Virais/genética
12.
Viruses ; 13(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: covidwho-1227073

RESUMO

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/sangue , COVID-19/genética , Ensaios de Triagem em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , RNA Viral/sangue , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
13.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1249580

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
14.
Sci Rep ; 11(1): 12676, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: covidwho-1275954

RESUMO

Regular PCR testing of nasopharyngeal swabs from symptomatic individuals for SARS-CoV-2 virus has become the established method by which health services are managing the COVID-19 pandemic. Businesses such as AstraZeneca have also prioritised voluntary asymptomatic testing to keep workplaces safe and maintain supply of essential medicines to patients. We describe the development of an internal automated SARS-CoV-2 testing programme including the transformative introduction of saliva as an alternative sample type.


Assuntos
Doenças Assintomáticas/epidemiologia , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/epidemiologia , Pandemias/prevenção & controle , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Saliva/virologia , Recursos Humanos , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , Nasofaringe/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos
15.
Biomed Res Int ; 2021: 6653950, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1263958

RESUMO

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Assuntos
COVID-19/diagnóstico , Proteínas do Envelope de Coronavírus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , 2-Propanol/química , Biomarcadores/análise , COVID-19/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Humanos , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Sci Rep ; 11(1): 11899, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1260951

RESUMO

The pandemic caused by SARS-CoV-2 resulted in increasing demands for diagnostic tests, leading to a shortage of recommended testing materials and reagents. This study reports on the performance of self-sampled alternative swabbing material (ordinary Q-tips tested against flocked swab and rayon swab), of reagents for classical RNA extraction (phenol/guanidine-based protocol against a commercial kit), and of intercalating dye-based one-step quantitative reverse transcription real-time PCRs (RT-qPCR) compared against the gold standard hydrolysis probe-based assays for SARS-CoV-2 detection. The study found sampling with Q-tips, RNA extraction with classical protocol and intercalating dye-based RT-qPCR as a reliable and comparably sensitive strategy for detection of SARS-CoV-2-particularly valuable in the current period with a resurgent and dramatic increase in SARS-CoV-2 infections and growing shortage of diagnostic materials especially for regions limited in resources.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/patogenicidade , Manejo de Espécimes , Teste para COVID-19/métodos , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/fisiologia , Manejo de Espécimes/métodos , Fatores de Tempo
17.
Virol J ; 18(1): 110, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: covidwho-1255943

RESUMO

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Proteínas do Envelope de Coronavírus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Limite de Detecção , Poliproteínas/genética , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Proteínas Virais/genética
18.
Methods Mol Biol ; 2277: 247-268, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080155

RESUMO

Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per µL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.


Assuntos
Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ácidos Nucleicos Livres/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Humanos , Leucócitos Mononucleares/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação
19.
Methods Mol Biol ; 2277: 345-356, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080161

RESUMO

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage which includes the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.


Assuntos
DNA Mitocondrial/análise , DNA Mitocondrial/efeitos da radiação , Eletroforese em Gel de Ágar/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pele/efeitos da radiação , Biomarcadores/análise , Dano ao DNA , DNA Mitocondrial/isolamento & purificação , Marcadores Genéticos , Humanos , Raios Ultravioleta/efeitos adversos
20.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061896

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
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