Simultaneous Detection of Influenza A, Influenza B, and Respiratory Syncytial Viruses and Subtyping of Influenza A H3N2 Virus and H1N1 (2009) Virus by Multiplex Real-Time PCR

Chen, Yu; Cui, Dawei; Zheng, Shufa; Yang, Shigui; Tong, James; Yang, De Ren; Fan, Jian Gao; Zhang, Jie; Liu, Bin; Li, Xuefen; Zhuge, Xiaoling; Ye, Bo; Zheng, Min; Mao, Weilin; Tan, Yajun; Xu, Guofan; Chen, Zhenjin; Chen, Nan; Li, Lanjuan

doi:10.1128/jcm.02184-10

Cited by 43 publications

(45 citation statements)

References 12 publications

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“…Estos primers y sondas TaqMan han sido considerados de publicaciones previas, e incluyen un control interno que es el gen humano GAPDH (14,21) . Los primers y sondas fueron sintetizadas por BioResearch (México).…”

Section: Diseño De Primers Y Sondasunclassified

Detección de virus influenza A, B y subtipos A (H1N1) pdm09, A (H3N2) por múltiple RT-PCR en muestras clínicas

Marcos-Carbajal

Huaringa

Rojas

et al. 2017

Rev Peru Med Exp Salud Publica

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RESUMENObjetivos. Estandarizar la técnica de reacción en cadena de la polimerasa en tiempo real (RT-PCR) múltiple para la detección de virus influenza A, B y tipificación de subtipos A (H1N1) pdm09, A (H3N2) en muestras clínicas. Materiales y métodos. Se analizaron 300 muestras de hisopado nasofaríngeo. Esta metodología fue estandarizada en dos pasos: la primera reacción detectó el gen de la matriz del virus de influenza A, gen de la nucleoproteína del virus influenza B y el gen GAPDH de las células huésped. La segunda reacción detectó el gen de la hemaglutinina de los subtipos A (H1N1) pandémico (pdm09) y A (H3N2). Resultados. Se identificaron 109 muestras positivas a influenza A y B, de las cuales 72 fueron positivas a influenza A (36 positivas a influenza A (H1N1) pdm09 y 36 positivos a influenza A (H3N2)) y 37 muestras positivas a influenza B. 191 fueron negativas a ambos virus mediante RT-PCR en tiempo real multiplex. Se encontró una sensibilidad y especificidad del 100% al analizar los resultados de ambas reacciones. El límite de detección viral fue del rango de 7 a 9 copias/µL por virus. Los resultados no mostraron ninguna reacción cruzada con otros virus tales como adenovirus, virus sincitial respiratorio, parainfluenza (1,2 y 3), metapneumovirus, subtipos A (H1N1) estacional, A (H5N2) y VIH. Conclusiones. La RT-PCR múltiple demostró ser una prueba muy sensible y específica para la detección de virus influenza A, B y subtipos A (H1N1, H3N2) y su uso puede ser conveniente en brotes estacionales. Palabras clave: Reacción en cadena de la polimerasa multiplex; Virus de la influenza A; Subtipo H1N1 del virus de la influenza A; Subtipo H3N2 del virus de la influenza A (fuente: DeCS BIREME). DETECTION OF INFLUENZA A, B AND SUBTYPES A (H1N1) pdm09, A (H3N2) VIRUSES BY MULTIPLE QRT-PCR IN CLINICAL SAMPLES ABSTRACTObjectives. To describe the clinical and epidemiological characteristics of patients diagnosed with epidermolysis bullosa (EB) at the Instituto Nacional de Salud (INSN) in Lima, Peru; a National Reference Center for this disease. Material and methods. Observational, descriptive and transversal study. We reviewed the clinical histories and laboratory tests of patients diagnosed with EB treated in INSN from 1993 to 2015. Results. 93 patients were registered. The average age was 7.9 ± 5.6 years; 53.8% (n = 50) were boys. Clinical forms corresponded to dystrophic EB with 41 (44.1%) cases, simple EB with 39 (41.9%) union EB cases with 8 (8.6%) and Kindler syndrome with 4 (4.3%) cases. The clinical form could not be identified in a case. A total of 48 cases (51.6%) came from Lima and Callao, and 45 cases (48.4%) from other provinces of the country. Extracutaneous manifestations involved gastrointestinal (44.1%), ocular (37.6%), odontogenic (87.1%), and nutritional (79.6%) involvement, as well as pseudosindactilia (16.1%). Chronic malnutrition (71.6%), acute malnutrition (17.6%) and anemia (62.4%) were found. Mortality corresponded to 6 cases (6.5%).Conclusions. 93 cases of EB were reported in INSN, the predomi...

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Section: Diseño De Primers Y Sondasunclassified

Detección de virus influenza A, B y subtipos A (H1N1) pdm09, A (H3N2) por múltiple RT-PCR en muestras clínicas

Marcos-Carbajal

Huaringa

Rojas

et al. 2017

Rev Peru Med Exp Salud Publica

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“…Influenza A (Flu A) virus is the most widespread pathogen causing human respiratory infections, which is historically associated with annual seasonal epidemics and influenza pandemics worldwide, such as those outbreaks in 1918, 1957, 1968, and 2009 (Cox and Subbarao 2000; Chen et al 2011; Bedford et al 2015). The human H1, H2, and H3 subtypes of the FluA viruses generally cause human infections.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Thus, the early detection and diagnosis of the suspected cases is the most effective measure to control and prevent further transmission of these FluA viruses (Chen et al 2011, 2013; Bedford et al 2015). Currently, high-throughput and multiplex molecular techniques (polymerase chain reaction, PCR) for the detection of Flu A virus subtypes have been rapidly developed in many laboratories, as previously described (Chen et al 2011; Kang et al 2014; World Health Organization 2013). Therefore, we established a multiplex real-time reverse transcription-PCR (rRT-PCR) assay allowing the simultaneous detection and discrimination of the universal Flu A strains, pandemic (H1N1) 2009, human H3N2 and reassortant avian H7N9 subtypes, and the Rnase P gene (RP) served as an internal control to monitor the quality of the clinical samples in one reaction tube.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay

et al. 2016

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Background Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube.MethodsClinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay.ResultsThe limitation of detection (LOD) of the multiplex assay was 5.4 × 10−2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10−2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10−2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples.ConclusionsThese findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

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“…All of these specimens were re-tested for influenza A virus, influenza B virus and respiratory syncytial virus (RSV) using an in-house RT-PCR multiplex assay [3]. Testing for other non-influenza or non-RSV respiratory viruses was not undertaken.…”

mentioning

confidence: 99%

Detection of influenza A(H3N2) clade 3C.2a viruses in patients with suspected mumps in British Columbia, Canada, during the 2014/15 influenza season

et al. 2015

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Simultaneous Detection of Influenza A, Influenza B, and Respiratory Syncytial Viruses and Subtyping of Influenza A H3N2 Virus and H1N1 (2009) Virus by Multiplex Real-Time PCR

Cited by 43 publications

References 12 publications

Detección de virus influenza A, B y subtipos A (H1N1) pdm09, A (H3N2) por múltiple RT-PCR en muestras clínicas

Detección de virus influenza A, B y subtipos A (H1N1) pdm09, A (H3N2) por múltiple RT-PCR en muestras clínicas

Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay

Detection of influenza A(H3N2) clade 3C.2a viruses in patients with suspected mumps in British Columbia, Canada, during the 2014/15 influenza season

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