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1.
Euro Surveill ; 25(39)2020 10.
Artigo em Inglês | MEDLINE | ID: covidwho-811370

RESUMO

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nucleoproteínas/genética , Pandemias , Pneumonia Viral/diagnóstico , Mutação Puntual , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Busca de Comunicante , Infecções por Coronavirus/virologia , Primers do DNA , Erros de Diagnóstico , Reações Falso-Negativas , Feminino , Genes Virais , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Nucleoproteínas/análise , Filogenia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Romênia , Doença Relacionada a Viagens , Proteínas Virais/análise
2.
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
3.
Euro Surveill ; 25(39)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33006300

RESUMO

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nucleoproteínas/genética , Pandemias , Pneumonia Viral/diagnóstico , Mutação Puntual , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Busca de Comunicante , Infecções por Coronavirus/virologia , Primers do DNA , Erros de Diagnóstico , Reações Falso-Negativas , Feminino , Genes Virais , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Nucleoproteínas/análise , Filogenia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Romênia , Doença Relacionada a Viagens , Proteínas Virais/análise
4.
Euro Surveill ; 25(39)2020 10.
Artigo em Inglês | MEDLINE | ID: covidwho-808986

RESUMO

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nucleoproteínas/genética , Pandemias , Pneumonia Viral/diagnóstico , Mutação Puntual , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Busca de Comunicante , Infecções por Coronavirus/virologia , Primers do DNA , Erros de Diagnóstico , Reações Falso-Negativas , Feminino , Genes Virais , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Nucleoproteínas/análise , Filogenia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Romênia , Doença Relacionada a Viagens , Proteínas Virais/análise
5.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 331-334, 2020 Jul 27.
Artigo em Chinês | MEDLINE | ID: mdl-32935503

RESUMO

With the rapid development of molecular biology, the isothermal amplification technique has been used for the nucleic acid detection of parasites and other pathogens due to its high efficiency and rapid and simple procedures, and has become an important tool to promote the field detection and control of parasitic diseases. Recombinase-aided isothermal amplification assay (RAA), a novel isothermal amplification technique, which is simple and easy to perform, rapid for field detection, no need for high-end equipment, and rapid field detection, may amplify the target gene fragments within 5 to 20 min under an isothermal condition (usually 37 to 42 ℃) and achieve a real-time observation of the amplification results. RAA has been successfully employed for the nucleic acid detection of a wide range of parasites and other pathogens to date, and has shown a high sensitivity and specificity. Notably, such an assay is suitable for the large-scale field detection in non-lab environments, and is therefore considered to have a potential value of application in rapid field detections.


Assuntos
Técnicas de Amplificação de Ácido Nucleico , Doenças Parasitárias , Parasitologia , Primers do DNA , Humanos , Doenças Parasitárias/diagnóstico , Doenças Parasitárias/prevenção & controle , Parasitologia/métodos , Recombinases , Sensibilidade e Especificidade
6.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 335-339, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32935504

RESUMO

OBJECTIVE: To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of Schistosoma mansoni. METHODS: The 121 bp highly-repeated sequence of S. mansoni was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of S. mansoni genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of S. japonicum, S. haematobium, Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity. RESULTS: A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of S. mansoni within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/µL using recombinant plasmids as templates and 0.1 fg/µL using S. mansoni genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from S. japonicum, S. haematobium, A. duodenale and C. sinensis. CONCLUSIONS: A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of S. mansoni.


Assuntos
Genes de Helmintos , Técnicas de Amplificação de Ácido Nucleico , Parasitologia , Schistosoma mansoni , Esquistossomose mansoni , Animais , Primers do DNA , Genes de Helmintos/genética , Parasitologia/métodos , Recombinases , Schistosoma mansoni/genética , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/parasitologia , Sensibilidade e Especificidade
7.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 340-344, 2020 Jul 02.
Artigo em Chinês | MEDLINE | ID: mdl-32935505

RESUMO

OBJECTIVE: To establish a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification (RAA) assay. METHODS: The 12S rRNA gene of E. granulosus was selected as the target gene, and the specific primers and fluorescent probes for RAA assay were designed, screened and synthesized to establish a fluorescent RAA assay for detection of E. granulosus. The sensitivity of the fluorescent RAA assay was evaluated using different copy numbers of target gene sequence-contained recombinant plasmids and various concentrations of E. granulosus genomic DNA as templates, and the specificity of the fluorescent RAA assay was evaluated using the genomic DNA from E. granulosus, E. multilocularis, Schistosoma japonicum, S. mansoni, Ancylostoma duodenale, Clonorchis sinensis, Taenia saginata, Spirometra mansoni and Taenia solium as templates. RESULTS: A fluorescent RAA assay was successfully established for detection of E. granulosus, which achieved specific amplification of E. granulosus genomic DNA within 20 min at 39 ℃. The lowest detection limit of the fluorescent RAA assay was 10 copies/µL of recombinant plasmids and 0.1 ng/µL E. granulosus genomic DNA, which exhibited a high sensitivity, and the fluorescent RAA assay was all negative for the genomic DNA from E. multilocularis, S. japonicum, S. mansoni, A. duodenale, C. sinensis, T. saginata, Spirometra mansoni and T. solium, which exhibited a high specificity. In addition, this fluorescent RAA assay successfully detected genomic DNA from E. granulosus cysts. CONCLUSIONS: A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of E. granulosus.


Assuntos
Equinococose , Echinococcus granulosus , Técnicas de Amplificação de Ácido Nucleico , Animais , Primers do DNA , Equinococose/diagnóstico , Echinococcus granulosus/genética , Recombinases , Sensibilidade e Especificidade
8.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(4): 350-354, 2020 Jul 03.
Artigo em Chinês | MEDLINE | ID: mdl-32935507

RESUMO

OBJECTIVE: To establish a recombinase-aided isothermal amplification (RAA) assay for the nucleic acid detection of Angiostrongylus cantonensis. METHODS: The internal transcribed spacer-1 (ITS1) gene sequence of A. cantonensis was used as the detection target sequence, and the specific primers and probes were designed and synthesized, followed by screening of the primers and probes with the highest specificity, to establish the basic and fluorescent RAA assay for nucleic acid detection of A. cantonensis. The sensitivity of the fluorescent RAA assay was evaluated by using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A. cantonensis as the template DNA samples, and the specificity of the fluorescent RAA assay was evaluated by using the genomic DNA from A. cantonensis, Schistosoma mansoni, Ascaris lumbricoides, Clonorchis sinensis, Echinococcus granulosus and Ancylostoma duodenale, as well as Pomacea canaliculata and Biomphalaria straminea snail tissues as the template DNA samples. RESULTS: A fluorescent RAA assay was successfully established for nucleic acid detection of A. cantonensis, which achieved real-time amplification of the specific DNA fragment of A. cantonensis within 20 min at 37 ℃. By using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A. cantonensis as the DNA templates, the lowest detection limits of the fluorescent RAA assay were 10 copies/µL of recombinant plasmids and 100 pg/µL of genomic DNA, respectively. The fluorescent RAA assay was negative for detection of the genomic DNA from A. cantonensis, S. mansoni, A. lumbricoides, C. sinensis, E. granulosus, A. duodenale, and P. canaliculata and B. straminea snail tissues. CONCLUSIONS: A simple, rapid fluorescent RAA assay has been successfully established, which has a high sensitivity and specificity for the nucleic acid detection of A. cantonensis.


Assuntos
Angiostrongylus cantonensis , Clonorchis sinensis , Técnicas de Amplificação de Ácido Nucleico , Parasitologia , Infecções por Strongylida , Angiostrongylus cantonensis/genética , Animais , Primers do DNA , Parasitologia/métodos , Recombinases , Sensibilidade e Especificidade , Infecções por Strongylida/diagnóstico
9.
Nat Commun ; 11(1): 4812, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: covidwho-791640

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
10.
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
11.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-781670

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
12.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946527

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
13.
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
14.
Genes (Basel) ; 11(8)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32806776

RESUMO

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Betacoronavirus/patogenicidade , Chlorocebus aethiops , Primers do DNA/normas , Feminino , Genoma Viral , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase/normas , Células Vero , Sequenciamento Completo do Genoma/normas
15.
Exp Parasitol ; 217: 107960, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32755552

RESUMO

Guinea worm Dracunculus medinensis causes debilitating disease in people and is subject to an ongoing global eradication programme. Research and controls are constrained by a lack of diagnostic tools. We developed a specific and sensitive LAMP method for detecting D. medinensis larval DNA in copepod vectors. We were able to detect a single larva in a background of field-collected copepods. This method could form the basis of a "pond-side test" for detecting potential sources of Guinea worm infection in the environment, in copepods, including in the guts of fish as potential transport hosts, enabling research, surveillance and targeting of control measures. The key constraint on the utility of this assay as a field diagnostic, is a lack of knowledge of variation in the temporal and spatial distribution of D. medinensis larvae in copepods in water bodies in the affected areas and how best to sample copepods to obtain a reliable diagnostic sample. These fundamental knowledge gaps could readily be addressed with field collections of samples across areas experiencing a range of worm infection frequencies, coupled with field and laboratory analyses using LAMP and PCR.


Assuntos
Copépodes/parasitologia , Dracunculus/isolamento & purificação , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Tanques/parasitologia , África , Animais , Sequência de Bases , Gatos , Copépodes/genética , Primers do DNA/química , DNA de Helmintos/isolamento & purificação , Vetores de Doenças , Cães , Dracunculus/genética , Humanos , Papio , Sensibilidade e Especificidade , Fatores de Tempo
16.
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
17.
PLoS One ; 15(8): e0236709, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790736

RESUMO

BACKGROUND: With the development of second-generation sequencing technology, more and more DNA sequence variations have been detected. Exon sequencing is the first choice for sequencing many cancer genes, and it can be better used to identify disease status by detecting gene variants. PCR sequence is an effective method to capture that sequence of an exon in the process of sequencing. Exon sequencing sequence contains PCR primer sequence, the correct position of the sequence can be determined by PCR primer sequence, which can be found in SNP, Indel mutation point by comparing the sequence of PCR primer sequence. RESULTS: In this paper, a matching algorithm based on the PCR primer sequence is proposed, which can effectively sequence the position of PCR primer sequence and find out the key position sequence. Then the sequencing sequence is sorted and the number of the same sequence is counted to reduce the matching times. Then, the sequenced sequence was matched with PCR primer sequence, so that the DNA position could be accurately matched and the variation in the sequenced sequence could be found more quickly. CONCLUSIONS: Compared with the traditional sequence matching method, PCR primer sequence matching method can match many sequences and find more variation. It also showed a high recall rate in the recall rate.


Assuntos
Algoritmos , Reação em Cadeia da Polimerase/métodos , DNA/química , DNA/genética , DNA/metabolismo , Primers do DNA/metabolismo , Éxons , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
18.
Genes (Basel) ; 11(8)2020 08 12.
Artigo em Inglês | MEDLINE | ID: covidwho-721491

RESUMO

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/métodos , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Betacoronavirus/patogenicidade , Chlorocebus aethiops , Primers do DNA/normas , Feminino , Genoma Viral , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase/normas , Células Vero , Sequenciamento Completo do Genoma/normas
19.
ACS Infect Dis ; 6(9): 2513-2523, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: covidwho-713585

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
20.
J Clin Virol ; 130: 104579, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: covidwho-694331

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
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