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

Cui, Dawei; Zhao, Dejian; Xie, Guoliang; Yang, Xianzhi; Huo, Zhaoxia; Zheng, Shufa; Yu, Fei; Chen, Yu

doi:10.1186/s40064-016-3733-9

Cited by 23 publications

(15 citation statements)

References 19 publications

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“…It represents a crucial strategy for parasite load determination in clinical samples, and allows the number of parasites to be normalized by the DNA quantity present in the sample [15]. For human patients, the RNAse P is a commonly used target as an internal control of reaction [56][57][58][59][60][61][62]. In Leishmania however, there are no studies, except the present one, describing the use of an internal control to quantify parasite load from human patient's samples.…”

Section: Discussionmentioning

confidence: 99%

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Filgueira¹,

Moreira²,

Cantanhêde³

et al. 2020

PLoS Negl Trop Dis

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Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.

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Section: Discussionmentioning

confidence: 99%

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Filgueira¹,

Moreira²,

Cantanhêde³

et al. 2020

PLoS Negl Trop Dis

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“…Virus isolation on embryonated chicken eggs and/or cell cultures is the gold-standard technique for IAV diagnosis; however, it is time-consuming and depends on virus viability [10]. Also, in the occurrence of co-infection, virus isolation could result in the selection of the dominant strain in the biological sample [23,24]. RT-PCR assay has been widely employed in diagnostic laboratories as a screening test for detection of influenza virus, using a conserved virus gene, matrix, or the nucleoprotein gene [25].…”

mentioning

confidence: 99%

Evaluation of two multiplex RT-PCR assays for detection and subtype differentiation of Brazilian swine influenza viruses

et al. 2020

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Influenza A virus (IAV) subtypes H1N1, H1N2, and H3N2 are endemic in swine herds in most pork producing countries; however, the viruses circulating in different geographic regions are antigenically and genetically distinct. In this sense, the availability of a rapid diagnostic assay to detect locally adapted IAVs and discriminate the virus subtype in clinical samples from swine is extremely important for monitoring and control of the disease. This study describes the development and validation of a multiplex RT-PCR assay for detection and subtyping of IAV from pigs. The analytical and diagnostic specificity of the assays was 100% (94.3-100.0, CI 95%), and the limit of detection was 10 −3 TCID 50 /mL. A total of 100 samples (IAV isolates and clinical specimens) were tested, and the virus subtype was determined for 80 samples (80%; 71.1-86.7, CI 95%). From these, 50% were H1N1, 22.5% were H1N2, and 7.5% were H3N2. Partial subtyping was determined for 8.75% samples (H1pdmNx and HxN2). Additionally, mixed infections with two virus subtypes (H1N2 + H3N2 and H1N1pdm + H1pdmN2; 2.5%) and reassortant viruses (H1pdmN2, 6.25%; and H1N1hu, 2.5%) were detected by the assay. A rapid detection of the most prevalent IAV subtypes and lineages in swine is provided by the assays developed here, improving the IAV diagnosis in Brazilian laboratories, and contributing to the IAV monitoring.

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“…We also evaluated the performance of the RT‐RAA assay in 342 avian clinical samples and compared it with the performance of published RT‐qPCR assays for H7 subtype AIV (Cui et al, ). The primers and probe for the RT‐qPCR assays are listed in Table .…”

Section: Methodsmentioning

confidence: 99%

“…The primers and probe for the RT‐qPCR assays are listed in Table . Detection was performed as described previously (Cui et al, ). Positive clinical samples were confirmed by conventional PCR and sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

Reverse‐transcription recombinase‐aided amplification assay for H7 subtype avian influenza virus

Wang

Huang

Liu

et al. 2019

Transbound Emerg Dis

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H7 subtype avian influenza virus infection is an emerging zoonosis in some Asian countries and an important avian disease worldwide. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. In this study, we developed a reverse‐transcription recombinase‐aided amplification assay for the detection of H7 subtype avian influenza virus. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. The assay showed no cross‐detection with Newcastle disease virus or infectious bronchitis virus, which are the other main respiratory viruses affecting birds. The analytical sensitivity was 102 RNA copies per reaction at a 95% probability level according to probit regression analysis, with 100% specificity. Compared with published reverse‐transcription quantitative real‐time polymerase chain reaction assays, the κ value of the reverse‐transcription recombinase‐aided amplification assay in 342 avian clinical samples was 0.988 (p < .001). The sensitivity for avian clinical sample detection was 100% (95%CI, 90.40%–100%), and the specificity was 99.96% (95%CI, 97.83%–99.98%). These results indicated that our reverse‐transcription recombinase‐aided amplification assay may be a valuable tool for detecting avian influenza H7 subtype virus.

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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

Cited by 23 publications

References 19 publications

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

Evaluation of two multiplex RT-PCR assays for detection and subtype differentiation of Brazilian swine influenza viruses

Reverse‐transcription recombinase‐aided amplification assay for H7 subtype avian influenza virus

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