Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex)

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction

BioTechniques ◽

10.2144/05382rr05 ◽

2005 ◽

Vol 38 (2) ◽

pp. 287-293 ◽

Cited By ~ 183

Author(s):

Van Luu-The ◽

Nathalie Paquet ◽

Ezequiel Calvo ◽

Jean Cumps

Keyword(s):

Real Time ◽

High Throughput ◽

Second Derivative ◽

Rt Pcr ◽

Pcr Method

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Rapid identification of H5 avian influenza virus in chicken throat swab specimens using microfluidic real-time RT-PCR

Analytical Methods ◽

10.1039/c3ay42126k ◽

2014 ◽

Vol 6 (8) ◽

pp. 2628 ◽

Cited By ~ 7

Author(s):

Ling Zhu ◽

Cancan Zhu ◽

Guoqing Deng ◽

Long Zhang ◽

Shumi Zhao ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Real Time ◽

Throat Swab ◽

Rapid Identification ◽

Rt Pcr

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Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

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Avian influenza and Newcastle disease virusindead chickens in markets in Dhaka, Bangladesh in 2011-2012

Bangladesh Veterinarian ◽

10.3329/bvet.v33i1.33308 ◽

2017 ◽

Vol 33 (1) ◽

pp. 8-15

Author(s):

LR Barman ◽

RD Sarker ◽

BC Das ◽

EH Chowdhury ◽

PM Das ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Newcastle Disease ◽

Rapid Test ◽

Type A ◽

Antigen Test ◽

Rt Pcr ◽

Test Kit ◽

Live Bird

A virological survey for avian influenza (AI) and Newcastle disease (ND) was conducted in two selected live bird markets (LBMs), namely Kaptan Bazar and Karwan Bazar in Dhaka city, Bangladesh from August 2011 to July 2012. A total of 513 dead chickens were collected. An immune-chromatographic rapid antigen test for Type A influenza virus and both conventional and real time RT-PCR were used for the detection and characterization of AI and ND viruses. All carcasses were first screened by the rapid antigen test kit and 93 were positive for Type A influenza virus. RT-PCR on a representative number of rapid antigen test positive samples (n = 24) confirmed the presence of Type A influenza virus and mostly H5 influenza virus (22 out of 24 tested samples). Influenza rapid test negative samples (n = 420) were subjected to routine necropsy. Heat stress, suffocation and physical injury were the most common cause of mortality (163 cases), followed by ND, suspected to be the cause of 85 deaths. On molecular investigation of these 85 samples, the presence of ND virus was confirmed in 59 and AI virus in 6; 15 were negative for both ND and AI viruses and 5 were unsuitable for investigation. Among the 59 ND confirmed cases 18 also contained AI virus. In summary, out of 513 carcasses 117 (22.81%) contained AI virus and 59 (11.50%) contained ND virus. Eighteen (3.51%) carcasses contained both AI and ND viruses. The findings suggest that both AI and ND should be considered as major threats to the poultry industry.Bangl. vet. 2016. Vol. 33, No. 1, 8-15

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Multiplex Detection of Rotavirus A, B, C, Norovirus GI and GII, Adenovirus, Astrovirus and Sapovirus in a Single PCR System in Stool Samples From Acute Diarrhea Children

10.21203/rs.3.rs-111216/v1 ◽

2020 ◽

Author(s):

Wei Li ◽

Weiwei Li ◽

Ran Tao ◽

Wenqing Xiang ◽

Mingming Zhou ◽

...

Keyword(s):

High Throughput ◽

Acute Diarrhea ◽

Cross Reaction ◽

Melting Curve Analysis ◽

Clinical Test ◽

Rt Pcr ◽

Rotavirus A ◽

Diarrhea Virus ◽

Intestinal Pathogens ◽

Stool Samples

Abstract Background: Early and accurate identification of infection viruses among children can benefit the personalized medical treatment and management, and reduce the future occurrence of serious symptoms. Thus, it is critical to develop a high-throughput multiplex real-time RT-PCR method to improve the accuracy and efficiency in routine clinical lab tests. Here, we developed a RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. Results: Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction through the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The detection limit was ranging from 1×102 to 1×105 copies/ml for each diarrhea virus. Conclusions: In conclusion, the RRCMC method is a suitable rapid clinical test for infection viruses, with the advantages of high-throughput, low cost, high sensitivity, and specificity.

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Expression profile of microRNAs in gastrointestinal stromal tumors revealed by high throughput quantitative RT-PCR microarray

World Journal of Gastroenterology ◽

10.3748/wjg.v21.i19.5843 ◽

2015 ◽

Vol 21 (19) ◽

pp. 5843-5855 ◽

Cited By ~ 13

Author(s):

Han-Xing Tong ◽

Yu-Hong Zhou ◽

Ying-Yong Hou ◽

Yong Zhang ◽

Yuan Huang ◽

...

Keyword(s):

Expression Profile ◽

High Throughput ◽

Gastrointestinal Stromal Tumors ◽

Rt Pcr ◽

Stromal Tumors ◽

Pcr Microarray

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High-throughput immunoassays for SARS-CoV-2, considerable differences in performance when comparing three methods

10.1101/2020.05.22.20106294 ◽

2020 ◽

Cited By ~ 1

Author(s):

Oskar Ekelund ◽

Kim Ekblom ◽

Sofia Somajo ◽

Johanna Pattison-Granberg ◽

Karl Olsson ◽

...

Keyword(s):

High Throughput ◽

Rapid Test ◽

Performance Criteria ◽

Clinical Use ◽

Rt Pcr ◽

Regulatory Authorities ◽

Flow Test ◽

Lateral Flow Test ◽

High Level ◽

Positive Results

Background: The recently launched high-throughput assays for the detection of antibodies against SARS-CoV-2 may change the managing strategies for the COVID-19 pandemic. This study aimed at investigating the performance of three high-throughput assays and one rapid lateral flow test relative to the recommended criteria defined by regulatory authorities. Methods: A total of 133 samples, including 100 pre-pandemic samples, 20 samples from SARS-CoV-2 RT-PCR positive individuals, and 13 potentially cross-reactive samples were analysed with SARS-CoV-2 IgG (Abbott), Elecsys Anti-SARS-CoV-2 (Roche), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) and 2019-nCOV IgG/IgM Rapid Test (Dynamiker Biotechnology Co). Results: All assays performed with a high level of specificity; however, only Abbott reached 100% (95% CI 96.3-100). The pre-pandemic samples analysed with Roche, DiaSorin and Dynamiker Biotechnology resulted in two to three false-positive results per method (specificity 96.9-98.0%). Sensitivity differed more between the assays, Roche exhibiting the highest sensitivity (100%, CI 83.9-100). The corresponding figures for Abbott, DiaSorin and Dynamiker Biotechnology were 85.0%, 77.8% and 75.0%, respectively. Conclusions: The results of the evaluated SARS-CoV-2 assays vary considerably as well as their ability to fulfil the performance criteria proposed by regulatory authorities. Introduction into clinical use in low-prevalent settings, should therefore, be made with caution.

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INFLUENZA INFLAMMATION BIOMARKERS FEATURES

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2018-3-67-73 ◽

2019 ◽

Vol 1 (3) ◽

pp. 67-73

Author(s):

T. P. Ospelnikova ◽

O. V. Morozova ◽

S. A. Andreeva ◽

E. I. Isaeva ◽

L. V. Kolodyazhnaya ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Influenza Virus ◽

Magnetic Beads ◽

Early Stage ◽

Influenza Infection ◽

Rt Pcr ◽

The Matrix ◽

Polarization Coefficient ◽

Th1 Immune Response

Aim. Analysis of inflammation biomarkers using reverse transcription with real time PCR (RT-PCR-RT) and multiplex immunofluorescent analysis xMAP with magnetic beads for the influenza infection. Materials and methods. Analysis of nasopharyngeal swabs, lymphocytes and blood sera of 10 patients with influenza and 10 donors was performed during the first 2 days of the disease by means of RT-PCR-RT and xMAP using the kit «37-plex» (BioRad). Results.The influenza virus A was revealed in 4 samples, the influenza virus B — in 6 swabs without mixed infections with other respiratory viruses. Analysis of the interferons (IFN) showed IFNα gene expression activation in patients’ lymphocytes but both the detection rate and the concentrations of IFNβ, IFNγ and IFNλ RNA were similar for patients and healthy donors. Among 37 inflammation biomarkers the concentrations of 7 proteins were enhanced including IFNα2, cytokines of TNF family (APRIL and BAFF), their soluble receptors sTNF-R1 and sTNF-R2, protein osteopontin and IL10. The concentrations of the complex of glycoprotein gp130 with the soluble receptor IL6 gp130/sIL-6Rβ and the matrix metalloprotease ММР-1 were reduced in patients’ sera. The polarization coefficient PI=[IL10]/[IFNγ]=0.53 for influenza samples suggested Th1 immune response. Conclusion. At the early stage of the influenza infection IFNα gene expression activation along with the induction of TNF family cytokines (APRIL and BAFF), their receptors (sTNF-R1 and sTNF-R2) and osteopontin as well as the inhibition of the complex gp130/sIL-6Rβ and metalloprotease ММР-1 were shown. Th1 immune response regulated by IL10 resulted in the recovery of the patients without complications.

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