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1.
Sci Rep ; 11(1): 16355, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381118

RESUMO

Rapid diagnostic tests are tools of paramount impact both for improving patient care and in antimicrobial management programs. Particularly in the case of respiratory infections, it is of great importance to quickly confirm/exclude the involvement of pathogens, be they bacteria or viruses, while obtaining information about the presence/absence of a genetic target of resistance to modulate antibiotic therapy. In this paper, we present our experiences with the use of the Biofire® FilmArray® Pneumonia Panel Plus (FAPP; bioMérieux; Marcy l'Etoile, France) to assess coinfection in COVID-19 patients. A total of 152 respiratory samples from consecutive patients were examined, and 93 (61%) were found to be FAPP positive, with the detection of bacteria and/or viruses. The patients were 93 males and 59 females with an average age of 65 years who were admitted to our hospital due to moderate/severe acute respiratory symptoms. Among the positive samples were 52 from sputum (SPU) and 41 from bronchoalveolar lavage (BAL). The most representative species was S. aureus (most isolates were mecA positive; 30/44, 62%), followed by gram-negative pathogens such as P. aeruginosa, K. pneumoniae, and A. baumannii. Evidence of a virus was rare. Cultures performed from BAL and SPU samples gave poor results. Most of the discrepant negative cultures were those in which FAPP detected pathogens with a microbial count ≤ 105 CFU/mL. H. influenzae was one of the most common pathogens lost by the conventional method. Despite the potential limitations of FAPP, which detects a defined number of pathogens, its advantages of rapid detection combined with predictive information regarding the antimicrobial resistance of pathogens through the detection of some relevant markers of resistance could be very useful for establishing empirical targeted therapy for the treatment of patients with respiratory failure. In the COVID era, we understand the importance of using antibiotics wisely to curb the phenomenon of antibiotic resistance.


Assuntos
COVID-19/complicações , Coinfecção , Testes Diagnósticos de Rotina , Infecções Respiratórias/complicações , Infecções Respiratórias/diagnóstico , Idoso , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia
2.
Trop Biomed ; 38(3): 283-288, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34362871

RESUMO

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.


Assuntos
Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase Multiplex , SARS-CoV-2/genética , Sequenciamento Completo do Genoma , Sequência de Bases , COVID-19 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos
3.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372564

RESUMO

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.


Assuntos
Bornaviridae/genética , Bornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , Doenças das Aves/virologia , Aves/genética , Aves/virologia , Primers do DNA/genética , Genoma Viral , Infecções por Mononegavirales/veterinária , Papagaios/genética , Papagaios/virologia , Passeriformes/genética , Passeriformes/virologia , Filogenia , RNA Viral/genética , Sequenciamento Completo do Genoma/métodos
4.
Arch Virol ; 166(9): 2551-2561, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34259914

RESUMO

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/genética , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/instrumentação , Monitoramento Epidemiológico , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/instrumentação , Cavidade Nasal/virologia , Nasofaringe/virologia , Fosfoproteínas/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Carga Viral
5.
Curr Issues Mol Biol ; 43(2): 728-748, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34287238

RESUMO

The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a severe threat to human health and the global economy and has resulted in overwhelming stress on health care systems worldwide. Despite the global health catastrophe, especially in the number of infections and fatalities, the COVID-19 pandemic has also revolutionized research and discovery with remarkable success in diagnostics, treatments, and vaccine development. The use of many diagnostic methods has helped establish public health guidelines to mitigate the spread of COVID-19. However, limited information has been shared about these methods, and there is a need for the scientific community to learn about these technologies, in addition to their sensitivity, specificity, and limitations. This review article is focused on providing insights into the major methods used for SARS-CoV-2 detection. We describe in detail the core principle of each method, including molecular and serological approaches, along with reported claims about the rates of false negatives and false positives, the types of specimens needed, and the level of technology and the time required to perform each test. Although this study will not rank or prioritize these methods, the information will help in the development of guidelines and diagnostic protocols in clinical settings and reference laboratories.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ensaio de Imunoadsorção Enzimática/métodos , Coloide de Ouro , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoensaio/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação
6.
ACS Appl Mater Interfaces ; 13(26): 30295-30305, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165969

RESUMO

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.


Assuntos
Hidrogéis/química , Reação em Cadeia da Polimerase Multiplex/métodos , Nanotubos de Carbono/química , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Compostos de Boro/química , Coronavirus/química , Primers do DNA/química , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Grafite/química , Vírus da Influenza A/química , Vírus da Doença de Newcastle/química , Estudo de Prova de Conceito , RNA Viral/química , Viroses/diagnóstico
7.
Mol Cell Probes ; 58: 101744, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34089805

RESUMO

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/virologia , Primers do DNA/genética , Sondas de DNA/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reprodutibilidade dos Testes , Ribonuclease P/genética , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética
8.
J Mol Diagn ; 23(9): 1078-1084, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34102313

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and has caused significant medical/socioeconomic impacts. Other than vaccination, effective public health measures, including contact tracing, isolation, and quarantine, is critical for deterring viral transmission, preventing infection progression and resuming normal activities. Viral transmission is affected by many factors, but the viral load and vitality could be among the most important ones. Although in vitro studies have indicated that the amount of virus isolated from infected individuals affects the successful rate of virus isolation, whether the viral load carried at the individual level would determine the transmissibility was unknown. We examined whether the cycle threshold (Ct) value, a measurement of viral load by RT-PCR assay, could differentiate the spreaders from the non-spreaders in a population of college students. Our results indicate that while at the population level the Ct value is lower, suggesting a higher viral load, in the symptomatic spreaders than that in the asymptomatic non-spreaders, there is a significant overlap in the Ct values between the two groups. Thus, Ct value, or the viral load, at the individual level could not predict the transmissibility. Instead, a sensitive method to detect the presence of virus is needed to identify asymptomatic individuals who may carry a low viral load but can still be infectious.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/transmissão , COVID-19/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Universidades/estatística & dados numéricos , COVID-19/epidemiologia , Portador Sadio/virologia , Busca de Comunicante , Feminino , Humanos , Louisiana/epidemiologia , Masculino , Nasofaringe/virologia , Saúde Pública , Quarentena , Estudos Retrospectivos , Estudantes/estatística & dados numéricos , Carga Viral , Adulto Jovem
9.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073924

RESUMO

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.


Assuntos
Exoma/genética , Doença de Gaucher/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , beta-Glucosidase/genética , Alelos , Variações do Número de Cópias de DNA , Família , Feminino , Doença de Gaucher/genética , Genótipo , Células HEK293 , Homozigoto , Humanos , Masculino , Mutação , Linhagem
10.
PLoS Biol ; 19(5): e3001236, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33961632

RESUMO

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/genética , Primers do DNA , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Poliproteínas/genética , Proteínas Virais/genética
11.
Nat Biomed Eng ; 5(7): 690-701, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33941896

RESUMO

DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show that low-frequency DNA variants can be detected via low-depth multiplexed NGS after their amplification, by a median of 300-fold, using polymerase chain reaction and rationally designed 'blocker' oligonucleotides that bind to the variants. Using an 80-plex NGS panel and a sequencing depth of 250×, we detected single nucleotide polymorphisms with a VAF of 0.019% and contamination in human cell lines at a VAF as low as 0.07%. With a 16-plex NGS panel covering 145 mutations across 9 genes involved in melanoma, we detected low-VAF mutations (0.2-5%) in 7 out of the 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be notably higher than previously recognized.


Assuntos
DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , DNA/genética , DNA/metabolismo , Bases de Dados Genéticas , Frequência do Gene , Biblioteca Gênica , Heterogeneidade Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Melanoma/genética , Melanoma/patologia , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Polimorfismo de Nucleotídeo Único
12.
Virol J ; 18(1): 89, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931105

RESUMO

BACKGROUND: A novel coronavirus (SARS-CoV-2) emerging has put global public health institutes on high alert. Little is known about the epidemiology and clinical characteristics of human coronaviruses infections in relation to infections with other respiratory viruses. METHODS: From February 2017 to December 2019, 3660 respiratory samples submitted to Zhejiang Children Hospital with acute respiratory symptoms were tested for four human coronaviruses RNA by a novel two-tube multiplex reverse transcription polymerase chain reaction assays. Samples were also screened for the occurrence of SARS-CoV-2 by reverse transcription-PCR analysis. RESULTS: Coronavirus RNAs were detected in 144 (3.93%) specimens: HCoV-HKU1 in 38 specimens, HCoV-NL63 in 62 specimens, HCoV-OC43 in 38 specimens and HCoV-229E in 8 specimens. Genomes for SARS-CoV-2 were absent in all specimens by RT-PCR analysis during the study period. The majority of HCoV infections occurred during fall months. No significant differences in gender, sample type, year were seen across species. 37.5 to 52.6% of coronaviruses detected were in specimens testing positive for other respiratory viruses. Phylogenic analysis identified that Zhejiang coronaviruses belong to multiple lineages of the coronaviruses circulating in other countries and areas. CONCLUSION: Common HCoVs may have annual peaks of circulation in fall months in the Zhejiang province, China. Genetic relatedness to the coronaviruses in other regions suggests further surveillance on human coronaviruses in clinical samples are clearly needed to understand their patterns of activity and role in the emergence of novel coronaviruses.


Assuntos
COVID-19/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Respiratórias/virologia , SARS-CoV-2/genética , Adolescente , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , COVID-19/complicações , COVID-19/genética , COVID-19/fisiopatologia , Criança , Pré-Escolar , China/epidemiologia , Coronavirus/genética , Coronavirus/isolamento & purificação , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/isolamento & purificação , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/isolamento & purificação , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/isolamento & purificação , Feminino , Hospitalização , Humanos , Lactente , Recém-Nascido , Masculino , Filogenia , Infecções Respiratórias/complicações , Infecções Respiratórias/etiologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética
13.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946969

RESUMO

The cytogenetic and molecular assessment of deletions, amplifications and rearrangements are key aspects in the diagnosis and therapy of cancer. Not only the initial evaluation and classification of the disease, but also the follow-up of the tumor rely on these laboratory approaches. The therapeutic choice can be guided by the results of the laboratory testing. Genetic deletions and/or amplifications directly affect the susceptibility or the resistance to specific therapies. In an era of personalized medicine, the correct and reliable molecular characterization of the disease, also during the therapeutic path, acquires a pivotal role. Molecular assays like multiplex ligation-dependent probe amplification and droplet digital PCR represent exceptional tools for a sensitive and reliable detection of genetic alterations and deserve a role in molecular oncology. In this manuscript we provide a technical comparison of these two approaches with the golden standard represented by fluorescence in situ hybridization. We also describe some relevant targets currently evaluated with these techniques in solid and hematologic tumors.


Assuntos
Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , Tecnologia Digital/métodos , Rearranjo Gênico , Proteínas de Neoplasias/genética , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Aberrações Cromossômicas , Emulsões , Determinação de Ponto Final/métodos , Fluorometria , Humanos , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas de Fusão Oncogênica/genética , Sensibilidade e Especificidade
14.
Curr Protoc ; 1(5): e145, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34004070

RESUMO

Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , Orofaringe/virologia , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Teste de Ácido Nucleico para COVID-19/economia , Humanos , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/análise , RNA Viral/isolamento & purificação
15.
Int J Infect Dis ; 107: 179-181, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33901651

RESUMO

OBJECTIVE: The aim of this study was to evaluate the QIAstat-Dx® Respiratory SARS-CoV-2 Panel (QIAstat-SARS-CoV-2), which is a closed, fully automated, multiplex polymerase chain reaction (PCR) assay that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 21 other pathogens that cause respiratory disease. METHODS: Nasopharyngeal swabs from patients with or suspected of having coronavirus disease 2019 were collected and tested at Bichat-Claude Bernard Hospital, Paris, France. Using the World Health Organisation-approved real-time-PCR assay developed by the Charité Institute of Virology as the reference, positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. RESULTS: In total, 189 negative and 88 positive samples were analyzed. QIAstat-SARS-CoV-2 had an NPA of 90.48% (95% confidence interval (CI), 85.37%, 94.26%) and a PPA of 94.32% (95% CI, 87.24%, 98.13%). Co-infections were detected by QIAstat-SARS-CoV-2 in 4/277 specimens. The methods exhibited comparable failure rates (23/307 [7.5%] vs. 6/298 [2.0%] for QIAstat-SARS-CoV-2 and reference methods, respectively). The turnaround time was shorter for QIAstat-SARS-CoV-2 compared with the reference method (difference in mean -14:30 h [standard error, 0:03:23; 95% CI, -14:37, -14:24]; P < 0.001). CONCLUSIONS: QIAstat-SARS-CoV-2 shows good agreement with the reference assay, providing faster and accurate results for detecting SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , SARS-CoV-2/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Estudos Retrospectivos , Adulto Jovem
16.
Forensic Sci Int Genet ; 53: 102511, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33831817

RESUMO

To support efforts in prosecuting wildlife crimes, we developed and validated a multiplex High-Resolution Melt (M-HRM) assay for the identification of proboscidean taxa commonly required to be identified or excluded in ivory seizures and forensic casework: Asian elephant (Elephas maximus), African elephant (Loxodonta spp.), mammoth (Mammuthus spp.), and mastodon (Mammut spp.). Five hundred and fifty (550) blood, tissue, and ivory samples from individuals of these 4 proboscidean taxa were used to develop and validate the 2 proboscidean-specific mitochondrial sites targeted by this assay. The 28-basepair (bp) 16S ribosomal RNA (rRNA) and 54-bp cytochrome b (Cytb) gene segments yield a combination of melt peaks that create composite melt profiles unique to each of the 4 proboscidean taxa. Wildlife forensic laboratories can use this sensitive, rapid, and cost-effective assay to assist efforts to combat the unlawful commercialization of proboscidean ivory and to stop the poaching crisis leading to the decline of these ivory-bearing species in the wild.


Assuntos
Crime , DNA/genética , Elefantes/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária , Animais , Conservação dos Recursos Naturais , Citocromos b/genética , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Especificidade da Espécie , Temperatura de Transição
17.
Gene ; 785: 145620, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33794327

RESUMO

Fritillariae cirrhosae bulbus, a well-known and precious medicinal and edible herb in China, causes remarkable effects on swelling and relieving cough, with fewer side effects than other congeneric medicine. It has been subject to various cheaper congeneric adulteration because of its high price and limited production. In this paper, a rapid, high throughput, sensitive and efficient technique was described for simultaneous identification of F. cirrhosae bulbus and its common adulterants by employing multiplex ligation-dependent probe amplification coupled with high-resolution melting (MLPA-HRM) curve assay in their internal transcribed spacer 1 (ITS1) regions. This assay was highly sensitive with a detection limit of 0.19 ng genomic DNA, and highly specific with no cross-reaction with common adulterants. Mixed sample analysis showed as low as 10% adulteration can be detected from F. cirrhosae bulbus in one MLPA-HRM reaction. Overall, the method described in this paper is well suited for detecting adulteration in F. cirrhosae bulbus.


Assuntos
Sondas de DNA , DNA de Plantas , Fritillaria , Reação em Cadeia da Polimerase Multiplex/métodos , Fritillaria/classificação , Fritillaria/genética , Desnaturação de Ácido Nucleico , Sensibilidade e Especificidade
18.
PLoS One ; 16(4): e0250942, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33914804

RESUMO

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex/normas , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
19.
J Virol Methods ; 293: 114147, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33812943

RESUMO

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is posing a great threat to global health and economy. Due to the lack of broad diagnostic setup, consistent reagent supply lines, and access to laboratory instruments and equipment, it is undoubtedly an enormous burden for developing countries to face the crisis. OBJECTIVES: To develop a cost-effective, reliable and sensitive multiplex assay for SARS-CoV-2 screening which would expand the testing capacities of a developing and low-income country like Bangladesh. STUDY DESIGN: Initially a singleplex and then a multiplex real-time reverse-transcriptase PCR assays were developed targeting 2 nucleocapsid genes of SARS-CoV-2, and the human RNase P gene as an internal control using laboratory-made mastermixes. Three sets of primer- probes were designed for each of the target genes and one set was optimized for the final reaction set-up. Limit of detection, cross-reactivity and reproducibility were checked in order to assess the sensitivity and specificity of the assays, and validation was done using clinical specimens. RESULTS: Clinical evaluation of the new assays using 240 nasopharyngeal swabs showed 100 % sensitivity, specificity, and accuracy in detecting SARS-CoV-2 infection in human. Equal efficiency and concordant results were observed between the singleplex and multiplex approaches. Notably, the kit was able to detect SARS-CoV-2 RNA at very low concentration upto 5 copies/reaction. CONCLUSION: This is the first locally developed multiplex rRT-PCR kit in Bangladesh providing rapid and low-cost screening of COVID-19 which would be valuable for infection prevention and clinical management in the perspective of Bangladesh.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , SARS-CoV-2/genética , Teste de Ácido Nucleico para COVID-19/economia , Humanos , Limite de Detecção , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
20.
J Virol Methods ; 293: 114149, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33839185

RESUMO

A multiplex real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed based on the same primer and probe sequences of an existing U.S. CDC Emergency Use authorized test panel, targeting SARS-CoV-2 N1, N2 and human RNase P genes in singleplex. Both singleplex and multiplex assays demonstrated linear dynamic ranges of 8 orders of magnitude and analytical limits of detection of 5 RNA transcript copies/reaction. Both assays showed 100 % agreement with 364 previously characterized clinical specimens (146 positive and 218 negative) for detection of SARS-CoV-2 RNA. To further increase testing throughput, 40 positive and 20 negative four-specimen pools were tested by the multiplex assay and showed 97.75 % and 100 % congruence with individual specimen tests, respectively. rRT-PCR assay multiplexing and sample pooling, individually or in combination, can substantially increase throughput of SARS-CoV-2 testing.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , SARS-CoV-2/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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