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1.
Biomed Res Int ; 2020: 7610678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029522

RESUMO

Background: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. Methods: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. Results: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. Conclusions: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Pneumonia Viral/diagnóstico , Sondas RNA/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
2.
Nat Commun ; 11(1): 4812, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968075

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Benchmarking , Técnicas de Laboratório Clínico/normas , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Primers do DNA/genética , Temperatura Alta , Humanos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade , Suécia/epidemiologia , Ensaio de Placa Viral/métodos
3.
J Clin Virol ; 130: 104579, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32795959

RESUMO

BACKGROUND: Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed. OBJECTIVES: To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials. MATERIAL AND METHODS: Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene. RESULTS: The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 µl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing. CONCLUSION: Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sistema Respiratório/virologia , Manejo de Espécimes/métodos , Betacoronavirus , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/análise , Sensibilidade e Especificidade , Fatores de Tempo
4.
ACS Infect Dis ; 6(9): 2513-2523, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32786273

RESUMO

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within 8 months of the outbreak, more than 10,000,000 cases of COVID-19 have been confirmed worldwide. Since human-to-human transmission occurs easily and the rate of human infection is rapidly increasing, sensitive and early diagnosis is essential to prevent a global outbreak. Recently, the World Health Organization (WHO) announced various primer-probe sets for SARS-CoV-2 developed at different institutions: China Center for Disease Control and Prevention (China CDC, China), Charité (Germany), The University of Hong Kong (HKU, Hong Kong), National Institute of Infectious Diseases in Japan (Japan NIID, Japan), National Institute of Health in Thailand (Thailand NIH, Thailand), and US CDC (USA). In this study, we compared the ability to detect SARS-CoV-2 RNA among seven primer-probe sets for the N gene and three primer-probe sets for the Orf1 gene. The results revealed that "NIID_2019-nCOV_N" from the Japan NIID and "ORF1ab" from China CDC represent a recommendable performance of RT-qPCR analysis for SARS-CoV-2 molecular diagnostics without nonspecific amplification and cross-reactivity for hCoV-229E, hCoV-OC43, and MERS-CoV RNA. Therefore, the appropriate combination of NIID_2019-nCOV_N (Japan NIID) and ORF1ab (China CDC) sets should be selected for sensitive and reliable SARS-CoV-2 molecular diagnostics.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Primers do DNA/genética , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Surtos de Doenças , Humanos , Pandemias , Patologia Molecular/métodos , Pneumonia Viral/diagnóstico , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos
5.
BMC Bioinformatics ; 21(1): 311, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677889

RESUMO

BACKGROUND: Polyploid organisms such as wheat complicate even the simplest of procedures in molecular biology. Whilst knowledge of genomic sequences in crops is increasing rapidly, the scientific community is still a long way from producing a full pan-genome for every species. Polymerase chain reaction and Sanger sequencing therefore remain widely used as methods for characterizing gene sequences in many varieties of crops. High sequence similarity between genomes in polyploids means that if primers are not homeologue-specific via the incorporation of a SNP at the 3' tail, sequences other than the target sequence will also be amplified. Current consensus for gene cloning in wheat is to manually perform many steps in a long bioinformatics pipeline. RESULTS: Here we present AutoCloner ( www.autocloner.com ), a fully automated pipeline for crop gene cloning that includes a free-to-use web interface for users. AutoCloner takes a sequence of interest from the user and performs a basic local alignment search tool (BLAST) search against the genome assembly for their particular polyploid crop. Homologous sequences are then compiled with the input sequence into a multiple sequence alignment which is mined for single-nucleotide polymorphisms (SNPs). Various combinations of potential primers that cover the entire gene of interest are then created and evaluated by Primer3; the set of primers with the highest score, as well as all possible primers at every SNP location, are then returned to the user for polymerase chain reaction (PCR). We have successfully used AutoCloner to clone various genes of interest in the Apogee wheat variety, which has no current genome sequence. In addition, we have successfully run the pipeline on ~ 80,000 high-confidence gene models from a wheat genome assembly. CONCLUSION: AutoCloner is the first tool to fully-automate primer design for gene cloning in polyploids, where previously the consensus within the wheat community was to perform this process manually. The web interface for AutoCloner provides a simple and effective polyploid primer-design method for gene cloning, with no need for researchers to download software or input any other details other than their sequence of interest.


Assuntos
Clonagem Molecular , Biologia Computacional/métodos , Primers do DNA/metabolismo , Poliploidia , Homologia de Sequência , Software , Triticum/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Primers do DNA/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética
6.
BMC Infect Dis ; 20(1): 550, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727378

RESUMO

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N8/genética , Influenza Aviária/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Neuraminidase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Aves/virologia , Primers do DNA/genética , Feminino , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
J Virol Methods ; 284: 113926, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650037

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70 %. Improved sensitivity is, therefore, urgently required. METHODS: We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances. RESULTS: The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2. CONCLUSION: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Humanos , Japão , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Estados Unidos
8.
J Virol Methods ; 284: 113937, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32659241

RESUMO

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Eletroforese Capilar/métodos , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , DNA Complementar/genética , Genoma Viral , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/diagnóstico , Sensibilidade e Especificidade
9.
Arch Virol ; 165(10): 2241-2247, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32681408

RESUMO

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 ℃ and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/µL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/µL for HPV16 and 100 copies/µL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.


Assuntos
DNA Viral/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Técnicas de Amplificação de Ácido Nucleico , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Adolescente , Adulto , Idoso , Colo do Útero/patologia , Colo do Útero/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/classificação , Papillomavirus Humano 18/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
10.
Arch Virol ; 165(10): 2335-2340, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32719956

RESUMO

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.


Assuntos
Infecções por Caliciviridae/virologia , Primers do DNA/química , Gastroenterite/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sapovirus/genética , Proteínas Estruturais Virais/genética , Sequência de Bases , Infecções por Caliciviridae/diagnóstico , Primers do DNA/genética , Fezes/virologia , Gastroenterite/diagnóstico , Expressão Gênica , Genótipo , Humanos , Tipagem Molecular/métodos , Filogenia , Sapovirus/classificação , Sapovirus/isolamento & purificação , Alinhamento de Sequência
11.
J Clin Virol ; 129: 104499, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32535397

RESUMO

BACKGROUND: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. METHODS: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. RESULTS: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction. CONCLUSIONS: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Sondas de Oligonucleotídeos/genética , Pneumonia Viral/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Humanos , Pandemias , RNA Viral/genética , Sensibilidade e Especificidade
12.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510493

RESUMO

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 °C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 5.


Assuntos
Ração Animal/microbiologia , Microbiologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Salmonella/genética , Salmonella/isolamento & purificação , Animais , Primers do DNA/genética , Limite de Detecção , Temperatura , Fatores de Tempo
13.
Food Chem ; 330: 127247, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32535319

RESUMO

Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.


Assuntos
Técnicas Biossensoriais , Primers do DNA/genética , Reação em Cadeia da Polimerase Multiplex , Plantas Geneticamente Modificadas
14.
Food Chem ; 324: 126821, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32361093

RESUMO

As large-scale planting of genetically modified (GM) crops increases, the development of a rapid and convenient method for on-site detection of GM crops is important. We combined the advantages of recombinase polymerase amplification (RPA) and fluorescence detection to establish a rapid, sensitive, specific, and simple detection platform for on-site detection of MON863 maize. Test samples were added directly to the platform after simple pre-treatment with a DNA extraction-free method. Results were obtained through real-time monitoring with a portable instrument, which facilitated sample-in/answer-out on-site detection. The entire detection process, including sample preparation, RPA and identification of amplification results, was accomplished in approximately 10 min. Furthermore, the detection was achieved with a simple and inexpensive portable device. This method has high potential for application in other fields requiring rapid detection of DNA targets, such as in field research, resource-limited areas, and science education.


Assuntos
Produtos Agrícolas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Primers do DNA/genética , Fluorescência , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/genética
15.
Biol Res ; 53(1): 21, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32410692

RESUMO

BACKGROUND: Liriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm. RESULTS: We performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79. CONCLUSIONS: The genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.


Assuntos
Genoma de Cloroplastos/genética , Genoma de Planta/genética , Liriodendron/genética , Polimorfismo Genético/genética , Alelos , Primers do DNA/genética , DNA de Plantas/genética , Genótipo , Repetições de Microssatélites , Sequenciamento Completo do Genoma
16.
Emerg Infect Dis ; 26(8)2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32396505

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10-1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Proteínas do Nucleocapsídeo/genética , Pneumonia Viral/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Biomarcadores/análise , Centers for Disease Control and Prevention, U.S. , Infecções por Coronavirus/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Fezes/virologia , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Escarro/virologia , Estados Unidos
17.
Nucleic Acids Res ; 48(12): e70, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32427335

RESUMO

Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA-Seq/métodos , Software , Primers do DNA/química , Primers do DNA/genética , Origem da Vida , RNA/química , RNA/genética
18.
Int J Food Microbiol ; 325: 108647, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32361480

RESUMO

Yeasts are one of the main organisms in the food industry and effective components of many ecosystems. The method for identifying and detecting certain yeast species or strains is a crucial step for the food industry and should be simple, reliable, fast, and inexpensive. In our study, inter-priming binding sites (iPBS) retrotransposon marker system was employed to elucidate the genetic variability at intraspecies and interspecies levels among 112 yeast strains belonging to eight species previously obtained from fermented foods. The molecular identification of yeast strains was firstly confirmed by sequencing the D1/D2 domain of the 26S rRNA. The eight selected retrotransposon-based primers produced 278 bands, all of which were polymorphic with an average of 34.75 polymorphic fragments per primer. The averages of polymorphism information contents and the resolving power values for the iPBS marker system were 0.23 and 10.11, respectively. The genetic parameters within each yeast species obtained from iPBS markers were observed as; the percentage of polymorphic loci for each species ranging from 19.23% to 71.21%, Nei's gene diversity from 0.085 to 0.228, while Shannon's information index values ranging from 0.125 to 0.349. The value of gene flow (0.09) and genetic variation among the populations (0.85) showed higher genetic variation among the species. UPGMA analyses demonstrated considerable genetic variability in the yeast strains, clustered them according to their species, and revealed the intraspecific variation. Each of the selected iPBS primer provided enough species-discrimination. Present evaluations suggest the utility of iPBS marker system to estimate the genetic variation of yeast strains. This study is a preliminary point for further studies on the identification methodology, and population genetics of yeast species having importance in the food industry with iPBS markers.


Assuntos
Retroelementos/genética , Saccharomycetales/genética , Fermento Seco/genética , Sítios de Ligação , Primers do DNA/genética , Ecossistema , Variação Genética/genética , Filogenia , Polimorfismo Genético/genética , Saccharomycetales/classificação
19.
Microb Biotechnol ; 13(4): 950-961, 2020 07.
Artigo em Inglês | MEDLINE | ID: covidwho-116666

RESUMO

The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/classificação , Betacoronavirus/genética , China , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Proteínas Virais/genética
20.
Microb Biotechnol ; 13(4): 950-961, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32333644

RESUMO

The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/classificação , Betacoronavirus/genética , China , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Pandemias , Pneumonia Viral/virologia , Sensibilidade e Especificidade , Proteínas Virais/genética
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