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1.
Virus Genes ; 56(3): 288-297, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32193781

RESUMO

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses timely makes it a powerful tool for public health emergency response. Third-generation sequencing (TGS) offers advantages in speed and length of detection over second-generation sequencing (SGS). Here, we presented the end-to-end workflows for both Oxford Nanopore MinION and Pacbio Sequel on a viral disease emergency event, along with Ion Torrent PGM as a reference. A specific pipeline for comparative analysis on viral genomes recovered by each platform was assembled, given the high errors of base-calling for TGS platforms. All the three platforms successfully identified and recovered at least 85% Norovirus GII genomes. Oxford Nanopore MinION spent the least sample-to-answer turnaround time with relatively low but enough accuracy for taxonomy classification. Pacbio Sequel recovered the most accurate viral genome, while spending the longest time. Overall, Nanopore metagenomics can rapidly characterize viruses, and Pacbio Sequel can accurately recover viruses. This study provides a framework for designing the appropriate experiments that are likely to lead to accurate and rapid virus emergency response.

2.
Cancer Cell ; 37(3): 403-419.e6, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-32183952

RESUMO

Natural killer/T cell lymphoma (NKTCL) is an aggressive and heterogeneous entity of non-Hodgkin lymphoma, strongly associated with Epstein-Barr virus (EBV) infection. To identify molecular subtypes of NKTCL based on genomic structural alterations and EBV sequences, we performed multi-omics study on 128 biopsy samples of newly diagnosed NKTCL and defined three prominent subtypes, which differ significantly in cell of origin, EBV gene expression, transcriptional signatures, and responses to asparaginase-based regimens and targeted therapy. Our findings thus identify molecular networks of EBV-associated pathogenesis and suggest potential clinical strategies on NKTCL.

3.
Int J Infect Dis ; 93: 224-230, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32045697

RESUMO

OBJECTIVES: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity. METHODS: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison. RESULTS: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%. CONCLUSIONS: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.

4.
J Med Virol ; 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31981228

RESUMO

Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses.

5.
Int J Infect Dis ; 86: 108-113, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31288091

RESUMO

OBJECTIVES: Pertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis. The purpose of this study was to develop a rapid, simple and sensitive diagnostic test for detecting this pathogen. METHODS: Here we present a recombinase aided amplification (RAA) assay incorporating competitive internal amplification control (IAC) to detect Bordetella pertussis using the DNA obtained by boiling. This assay was performed in a single closed tube at 39°C within 30min. A total of 115 clinical samples suspected of pertussis were collected and tested by the internally controlled RAA assay using both extracted DNA with the commercial kit and the DNA obtained by boiling. For comparison, the real-time PCR (RT-PCR) was also performed with DNA extraction in parallel. RESULTS: The sensitivity of the internally controlled RAA assay was 101 copies or 10CFU/ml per reaction in detecting plasmid DNA or B. pertussis strain. The optimum concentration of the IAC plasmid was determined to be 100 copies, and the introduction of IAC effectively reduced the occurrence of false negatives. Compared to the RT-PCR, RAA results with DNA extraction obtained 100% sensitivity and specificity, and the RAA results with heat-treated DNA showed 85.96% sensitivity and 100% specificity. CONCLUSION: With the advantages of 45min turn-around time and simple steps of DNA purification, this assay could become a useful diagnostic tool for Bordetella pertussis detection and is potentially suitable for point-of-care identification to guide prompt clinical treatment.


Assuntos
Bordetella pertussis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases , Coqueluche/diagnóstico , Bordetella pertussis/genética , Criança , Pré-Escolar , DNA Bacteriano/isolamento & purificação , Feminino , Temperatura Alta , Humanos , Lactente , Masculino , Técnicas de Amplificação de Ácido Nucleico/normas , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Sensibilidade e Especificidade
6.
Virol J ; 16(1): 86, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262315

RESUMO

BACKGROUND: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control. METHODS: We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min. RESULTS: The analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol. CONCLUSIONS: We provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.


Assuntos
Adenovírus Humanos/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Recombinases/genética , Adenovírus Humanos/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Infecções Respiratórias/epidemiologia , Sensibilidade e Especificidade , Sorogrupo , Temperatura
7.
Biomed Environ Sci ; 32(5): 357-362, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31217052

RESUMO

OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION: A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , Vírus da Encefalite Transmitidos por Carrapatos/genética
8.
Biomed Environ Sci ; 32(5): 363-370, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31217053

RESUMO

OBJECTIVE: People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus (YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice. METHODS: In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV-negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice. RESULTS: The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus (89.58%), Epstein-Barr virus (55.21%), hepatitis C virus (34.38%), rhinovirus (28.13%), hepatitis A virus (20.83%), coxsackievirus (10.42%), Ebola virus (8.33%), hepatitis E virus (8.33%), lyssavirus (4.17%), leptospirosis (4.17%), chikungunya virus (2.08%), Crimean-Congo hemorrhagic fever virus (1.04%), and hepatitis B virus (1.04%). CONCLUSION: The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.


Assuntos
Febre/virologia , Icterícia/virologia , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Febre/epidemiologia , Humanos , Icterícia/epidemiologia , Masculino , Análise de Sequência , Serra Leoa/epidemiologia , Adulto Jovem
9.
BMC Infect Dis ; 19(1): 229, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30836947

RESUMO

BACKGROUND: Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. METHODS: Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). RESULTS: A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. CONCLUSIONS: We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.


Assuntos
Vírus da Hepatite B/isolamento & purificação , Hepatite B/diagnóstico , Técnicas de Amplificação de Ácido Nucleico , Recombinases/metabolismo , Adulto , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Feminino , Hepatite B/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
10.
Arch Virol ; 164(1): 63-68, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30255299

RESUMO

Some serotypes of enterovirus (EV) may lead to transient and symptomatic gastrointestinal infections while others are commensal residents of the human gut. To determine whether certain EV types are more often associated with diarrhea, we conducted a preliminary study on the prevalence of EV serotypes and common diarrhea viruses in fecal samples of diarrhea children and healthy controls. EV was tested with one step nest polymerase chain reaction and typed by direct sequencing while common causative diarrhea viruses rotavirus (RV), norovirus (NoV), adenovirus (AdV), bocavirus (HBoV), and astrovirus (AstV) were screened with multiplex PCR assays. Human Rhinovirus (HRV) and human EVs that were present in both groups were further quantified and their odds ratios (OR) were calculated. Enteric pathogens were detected in 89 (32.6%) of 273 children with diarrhea and included human EVs (51, 18.68%), HRV (32, 11.72%), RV (38, 13.92%), AdV (24, 8.79%), NoVGII (16, 8.79%), HBoV (8, 2.93%) and AstV (3, 1.09%). Potential enteric pathogens were found in 25 (6.93%) of 361 healthy controls and included human EV (59, 16.34%), HRV (8, 2.22%), RV (1, 0.28%), NoVGII (5, 1.39%), AstV (2, 0.55%), AdV (16, 4.43%) and HBoV (1, 0.28%). In addition, EV71, echovirus 3,9,14,25 and coxsackievirus A14 existed in healthy controls only, while HRV, echovirus11,18, coxsackievirus A2,4,6 and B2,4 were found in both patients and healthy controls. OR assessment confirmed a strong association of HRV (P < 0.001) and a weak one for echovirus 11 and coxsackievirus A6 with diarrhea (P > 0.05). Our results indicate the diversity of EV serotypes in diarrhea and healthy control groups varies, and the potential etiological role of HRV in diarrhea.


Assuntos
Diarreia/virologia , Infecções por Enterovirus/virologia , Enterovirus/isolamento & purificação , Estudos de Casos e Controles , Pré-Escolar , Fezes/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino
11.
Diagn Microbiol Infect Dis ; 93(2): 101-106, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30266400

RESUMO

Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid--based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02 × 10-1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.


Assuntos
Oligonucleotídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Primers do DNA/genética , Humanos , Limite de Detecção , Desnaturação de Ácido Nucleico , RNA Viral/análise , RNA Viral/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação
12.
Biomed Environ Sci ; 32(12): 926-929, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31918798

RESUMO

West Nile virus (WNV) causes West Nile fever and West Nile encephalitis. Because infection by WNV creates serious public health problems, its simple, rapid, and visual detection is very important in clinical practice, especially in resource-limited laboratories. We have developed a rapid, specific, and highly sensitive internally controlled reverse transcription recombinase-aided amplification (RTRAA) assay to detect WNV, using both real-time fluorescence and the lateral flow dipstick (LFD) at 39.0 °C for 30 min. The analytical sensitivity of the RT-RAA assay was 10 plasmid copies and 1.6 pfu per reaction with real-time fluorescence, and 1,000 plasmid copies per reaction with the LFD. No crossreaction with other control viruses was observed. Compared with the RT-qPCR assay, the RT-RAA assay demonstrated 100% sensitivity and 100% specificity for WNV.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismo , Transcrição Reversa , Vírus do Nilo Ocidental/isolamento & purificação , Fatores de Tempo , Vírus do Nilo Ocidental/genética
13.
Virol J ; 16(1): 166, 2019 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-31888694

RESUMO

BACKGROUND: Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. METHODS: Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. RESULTS: The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. CONCLUSIONS: The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.


Assuntos
Enterovirus Humano A/isolamento & purificação , Enterovirus/isolamento & purificação , Doença de Mão, Pé e Boca/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Enterovirus/genética , Enterovirus Humano A/genética , Humanos , Sensibilidade e Especificidade
14.
Virol J ; 15(1): 167, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30376870

RESUMO

BACKGROUND: Respiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment. RESULTS: A locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay. CONCLUSION: mOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.


Assuntos
Metapneumovirus/isolamento & purificação , Infecções por Paramyxoviridae/diagnóstico , Infecções por Picornaviridae/diagnóstico , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Rhinovirus/isolamento & purificação , Doença Aguda , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Metapneumovirus/genética , Reação em Cadeia da Polimerase Multiplex , Nasofaringe/virologia , Reprodutibilidade dos Testes , Vírus Sincicial Respiratório Humano/genética , Rhinovirus/genética , Sensibilidade e Especificidade
15.
BMC Infect Dis ; 18(1): 450, 2018 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-30176819

RESUMO

BACKGROUND: Diarrhea is a major source of morbidity and mortality among young children in low-income and middle-income countries. Human adenoviruses (HAdV), particular HAdV species F (40, 41) has been recognized as important causal pathogens, however limited data exist on molecular epidemiology of other HAdV associated with acute gastroenteritis. METHODS: In the present preliminary study, we performed a case-control study involving 273 children who presented diarrheal disease and 361 healthy children matched control in Children's hospital of Hebei Province (China) to investigate the relationship between non-enteric HAdV and diarrhea. HAdV were detected and quantified using quantitative real-time PCR (qPCR) and serotyped by sequencing and phylogenetic analysis. Odds ratio (OR) was used to assess the risk factor of HAdV. RESULTS: HAdV were detected in 79 (28.94%) of 273 children with diarrhea including 7 different serotypes (HAdV 40, 41, 3, 2,1,5 and 57) with serotypes 40, 41 and 3 being the most dominant and in 26 (7.20%) of 361 healthy children containing 9 serotypes (HAdV 40, 41, 3, 2,1,5,57,6 and 31). A majority (91.14%) of HAdV positives occurred in diarrhea children and 65.38% in controls< 3 years of age. No significant difference in the viral load was found between case and control groups or between Ad41-positive patients and healthy controls. In addition to HAdV 40 and 41, HAdV 3 was also associated with diarrhea (OR = 17.301, adjusted OR = 9.205, p < 0.001). CONCLUSIONS: Our results demonstrate a high diversity of HAdV present among diarrhea and healthy children and implicate that non-enteric HAdV3 may lead to diarrhea.


Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , Diarreia/epidemiologia , Diarreia/virologia , Infecções por Adenovirus Humanos/complicações , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , China/epidemiologia , Feminino , Gastroenterite/epidemiologia , Gastroenterite/virologia , Humanos , Lactente , Masculino , Epidemiologia Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Sorotipagem , Carga Viral
16.
Virol J ; 15(1): 81, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29716642

RESUMO

BACKGROUND: Human adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive. METHODS: In the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated. RESULTS: The analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138). CONCLUSION: The proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.


Assuntos
Adenovírus Humanos/genética , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/isolamento & purificação , Criança , Pré-Escolar , Humanos , Lactente , Tipagem Molecular/instrumentação , Tipagem Molecular/normas , Reação em Cadeia da Polimerase Multiplex/normas , Nasofaringe/virologia , Variações Dependentes do Observador , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Sorogrupo
17.
Biomed Environ Sci ; 31(4): 272-279, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29773090

RESUMO

OBJECTIVE: Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. METHODS: A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. RESULTS: A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. CONCLUSION: The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Viroses/diagnóstico , Viroses/virologia , Vírus/isolamento & purificação , Sequência de Bases , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase em Tempo Real
18.
Arch Virol ; 163(6): 1455-1461, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29429036

RESUMO

Hand, foot and mouth disease (HFMD) is a serious public health problem, and coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) are two of the major causative pathogens, in addition to enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). A simple and rapid reverse transcription recombinase-aided amplification assay (RT-RAA) was developed for the detection of CVA10 and CVA6 in this study. The analytical sensitivity for detection of CVA10 and CVA6 at 95% probability by probit regression analysis was 35 copies per reaction and 38 copies per reaction, respectively, with 100% specificity. Compared with commercial RT-qPCR assays, when testing 455 fecal specimens, the kappa value of the RT-RAA assay for CVA10 and CVA6 was 0.920 (p < 0.001) and 0.952 (p < 0.001), respectively. Moreover, four samples that were positive for CVA10 and five that were positive for CVA6 by RT-RAA but negative by RT-qPCR were further determined to be true positives. These results demonstrate that the proposed RT-RAA assays are very valuable tools for the detection of CVA10 and CVA6 and have potential for use in resource-limited settings.


Assuntos
Enterovirus/genética , Doença de Mão, Pé e Boca/diagnóstico , RNA Viral/genética , Recombinases/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Criança , Pré-Escolar , Primers do DNA/química , Primers do DNA/genética , Enterovirus/classificação , Enterovirus/isolamento & purificação , Fezes/virologia , Feminino , Doença de Mão, Pé e Boca/virologia , Humanos , Lactente , Masculino , Plasmídeos/química , Plasmídeos/metabolismo , Recombinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade
19.
Can J Microbiol ; 64(4): 223-230, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29351385

RESUMO

A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/genética , Vibrioses/diagnóstico , Vibrioses/microbiologia , Vibrio parahaemolyticus/genética , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/química , Primers do DNA/genética , DNA Bacteriano/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
20.
Diagn Microbiol Infect Dis ; 90(3): 181-185, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29273481

RESUMO

The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10-8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples.


Assuntos
Líquido Cefalorraquidiano/virologia , Encefalite/diagnóstico , Enterovirus Humano A/genética , Infecções por Enterovirus/diagnóstico , Meningite Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pré-Escolar , Encefalite/virologia , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/virologia , Fezes/virologia , Humanos , Meningite Viral/virologia , RNA Viral/genética , Sensibilidade e Especificidade
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