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  2. AU="Marcotullio, Peter J."

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  1. Artikel ; Online: Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2.

    Tombuloglu, Huseyin / Sabit, Hussein / Al-Suhaimi, Ebtesam / Al Jindan, Reem / Alkharsah, Khaled R

    PloS one

    2021  Band 16, Heft 4, Seite(n) e0250942

    Abstract: ... reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 ... to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used ... The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues ...

    Abstract The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.
    Mesh-Begriff(e) COVID-19/diagnosis ; COVID-19/virology ; COVID-19 Nucleic Acid Testing/methods ; COVID-19 Nucleic Acid Testing/standards ; Humans ; Limit of Detection ; Multiplex Polymerase Chain Reaction/methods ; Multiplex Polymerase Chain Reaction/standards ; RNA, Viral/analysis ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; SARS-CoV-2/isolation & purification ; Sensitivity and Specificity
    Chemische Substanzen RNA, Viral
    Sprache Englisch
    Erscheinungsdatum 2021-04-29
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't ; Validation Study
    ZDB-ID 2267670-3
    ISSN 1932-6203 ; 1932-6203
    ISSN (online) 1932-6203
    ISSN 1932-6203
    DOI 10.1371/journal.pone.0250942
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  2. Artikel ; Online: Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2.

    Huseyin Tombuloglu / Hussein Sabit / Ebtesam Al-Suhaimi / Reem Al Jindan / Khaled R Alkharsah

    PLoS ONE, Vol 16, Iss 4, p e

    2021  Band 0250942

    Abstract: ... reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 ... to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used ... The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues ...

    Abstract The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.
    Schlagwörter Medicine ; R ; Science ; Q
    Sprache Englisch
    Erscheinungsdatum 2021-01-01T00:00:00Z
    Verlag Public Library of Science (PLoS)
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  3. Artikel ; Online: Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection.

    Zhen, Wei / Berry, Gregory J

    The Journal of molecular diagnostics : JMD

    2020  Band 22, Heft 12, Seite(n) 1367–1372

    Abstract: This research describes the development of a new multiplex real-time RT-PCR test for detection ... of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike ... surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit ...

    Abstract This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC assay, with a final LOD of 1301 ± 13 genome equivalents/mL for the Northwell Health Laboratories laboratory-developed test (NWHL LDT) versus 1249 ± 14 genome equivalents/mL for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared with the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results show that the NWHL LDT multiplex assay performs as well as the modified CDC assay but is more efficient and cost-effective and can be used as a diagnostic assay and for epidemiologic surveillance and clinical management of SARS-CoV-2.
    Mesh-Begriff(e) Betacoronavirus/genetics ; Betacoronavirus/isolation & purification ; COVID-19 ; COVID-19 Testing ; COVID-19 Vaccines ; Clinical Laboratory Techniques ; Coronavirus Infections/diagnosis ; DNA Primers/genetics ; Humans ; Limit of Detection ; Multiplex Polymerase Chain Reaction/methods ; Pandemics ; Pneumonia, Viral/diagnosis ; SARS-CoV-2 ; Sensitivity and Specificity ; Spike Glycoprotein, Coronavirus/genetics
    Chemische Substanzen COVID-19 Vaccines ; Covid-19 aAPC vaccine ; DNA Primers ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
    Schlagwörter covid19
    Sprache Englisch
    Erscheinungsdatum 2020-09-19
    Erscheinungsland United States
    Dokumenttyp Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2000060-1
    ISSN 1943-7811 ; 1525-1578
    ISSN (online) 1943-7811
    ISSN 1525-1578
    DOI 10.1016/j.jmoldx.2020.09.004
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  4. Artikel ; Online: Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection

    Zhen, Wei / Berry, Gregory J.

    The Journal of Molecular Diagnostics

    2020  Band 22, Heft 12, Seite(n) 1367–1372

    Schlagwörter Pathology and Forensic Medicine ; Molecular Medicine ; covid19
    Sprache Englisch
    Verlag Elsevier BV
    Erscheinungsland us
    Dokumenttyp Artikel ; Online
    ZDB-ID 2000060-1
    ISSN 1943-7811 ; 1525-1578
    ISSN (online) 1943-7811
    ISSN 1525-1578
    DOI 10.1016/j.jmoldx.2020.09.004
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  5. Artikel: Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection

    Zhen, Wei / Berry, Gregory J

    J. mol. diagn

    Abstract: This research describes the development of a new multiplex real-time RT-PCR test for detection ... of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike ... surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit ...

    Abstract This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC assay, with a final LOD of 1301 ± 13 genome equivalents/mL for the Northwell Health Laboratories laboratory-developed test (NWHL LDT) versus 1249 ± 14 genome equivalents/mL for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared with the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results show that the NWHL LDT multiplex assay performs as well as the modified CDC assay but is more efficient and cost-effective and can be used as a diagnostic assay and for epidemiologic surveillance and clinical management of SARS-CoV-2.
    Schlagwörter covid19
    Verlag WHO
    Dokumenttyp Artikel
    Anmerkung WHO #Covidence: #779293
    Datenquelle COVID19

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  6. Buch ; Online: Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection.

    Zhen, W. / Berry, G. J.

    Journal Articles

    2020  

    Abstract: This research describes the development of a new multiplex real-time RT-PCR test for detection ... of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike ... surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit ...

    Abstract This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC assay, with a final LOD of 1301 ± 13 genome equivalents/mL for the Northwell Health Laboratories laboratory-developed test (NWHL LDT) versus 1249 ± 14 genome equivalents/mL for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared with the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results show that the NWHL LDT multiplex assay performs as well as the modified CDC assay but is more efficient and cost-effective and can be used as a diagnostic assay and for epidemiologic surveillance and clinical management of SARS-CoV-2.
    Schlagwörter Pathology ; covid19
    Thema/Rubrik (Code) 610
    Erscheinungsdatum 2020-01-01T08:00:00Z
    Verlag Donald and Barbara Zucker School of Medicine Academic Works
    Erscheinungsland us
    Dokumenttyp Buch ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  7. Artikel ; Online: Development of multiplex allele-specific RT-qPCR assays for differentiation of SARS-CoV-2 Omicron subvariants.

    Li, Jianguo / Cheng, Ruiling / Bian, Zixin / Niu, Jiahui / Xia, Juan / Mao, Guoli / Liu, Hulong / Wu, Changxin / Hao, Chunyan

    Applied microbiology and biotechnology

    2024  Band 108, Heft 1, Seite(n) 35

    Abstract: ... multiplex RT-qPCR reactions was applied to determine if a mutation occurs or not. SARS-CoV-2 subvariants and ... whole genome sequencing is required. Allele-specific reverse transcriptase real time RT-PCR (RT-qPCR ... successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 5 ...

    Abstract Quick differentiation of current circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmissions. However, the widely applied gene sequencing method is time-consuming and costly especially when facing recombinant variants, because a large part or whole genome sequencing is required. Allele-specific reverse transcriptase real time RT-PCR (RT-qPCR) represents a quick and cost-effective method for SNP (single nucleotide polymorphism) genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 5 multiplex allele-specific RT-qPCR assays targeting 20 key mutations for quick differentiation of the Omicron subvariants (BA.1 to BA.5 and their descendants) and the recombinant variants (XBB.1 and XBB.1.5). Two parallel multiplex RT-qPCR reactions were designed to separately target the prototype allele and the mutated allele of each mutation in the allele-specific RT-qPCR assay. Optimal annealing temperatures, primer and probe dosage, and time for annealing/extension for each reaction were determined by multi-factor and multi-level orthogonal test. The variation of Cp (crossing point) values (ΔCp) between the two multiplex RT-qPCR reactions was applied to determine if a mutation occurs or not. SARS-CoV-2 subvariants and related recombinant variants were differentiated by their unique mutation patterns. The developed multiplex allele-specific RT-qPCR assays exhibited excellent analytical sensitivities (with limits of detection (LoDs) of 1.47-18.52 copies per reaction), wide linear detection ranges (10
    Mesh-Begriff(e) Humans ; Alleles ; COVID-19/diagnosis ; Reproducibility of Results ; SARS-CoV-2/genetics
    Sprache Englisch
    Erscheinungsdatum 2024-01-06
    Erscheinungsland Germany
    Dokumenttyp Journal Article
    ZDB-ID 392453-1
    ISSN 1432-0614 ; 0171-1741 ; 0175-7598
    ISSN (online) 1432-0614
    ISSN 0171-1741 ; 0175-7598
    DOI 10.1007/s00253-023-12941-2
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  8. Artikel ; Online: Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV.

    Althobaiti, Atheer / Hamdan, Kareem / Sobhy, Mohamed A / Rawas, Renad / Takahashi, Masateru / Artyukh, Olga / Tehseen, Muhammad

    Journal of visualized experiments : JoVE

    2023  , Heft 201

    Abstract: ... we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT ... Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and ... qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV ...

    Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public's health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.
    Mesh-Begriff(e) Humans ; Middle East Respiratory Syndrome Coronavirus/genetics ; SARS-CoV-2/genetics ; Influenza, Human/diagnosis ; COVID-19/diagnosis ; RNA ; Sensitivity and Specificity
    Chemische Substanzen RNA (63231-63-0)
    Sprache Englisch
    Erscheinungsdatum 2023-11-10
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Video-Audio Media
    ZDB-ID 2259946-0
    ISSN 1940-087X ; 1940-087X
    ISSN (online) 1940-087X
    ISSN 1940-087X
    DOI 10.3791/65822
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  9. Artikel ; Online: Development and validation of RT-PCR assays for testing for SARS-CoV-2.

    Pabbaraju, Kanti / Wong, Anita A / Douesnard, Mark / Ma, Raymond / Gill, Kara / Dieu, Paul / Fonseca, Kevin / Zelyas, Nathan / Tipples, Graham A

    Journal of the Association of Medical Microbiology and Infectious Disease Canada = Journal officiel de l'Association pour la microbiologie medicale et l'infectiologie Canada

    2021  Band 6, Heft 1, Seite(n) 16–22

    Abstract: ... that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase ... time RT-PCR assays early in the pandemic was essential to provide valuable time to local health ... coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis ...

    Abstract Background: The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays.
    Methods: We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2-specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control.
    Results: These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention.
    Conclusions: The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.
    Sprache Englisch
    Erscheinungsdatum 2021-05-03
    Erscheinungsland Canada
    Dokumenttyp Journal Article
    ISSN 2371-0888
    ISSN (online) 2371-0888
    DOI 10.3138/jammi-2020-0026
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  10. Artikel ; Online: Development of multiplex RT-ddPCR assays for detection of SARS-CoV-2 and other common respiratory virus infections.

    Leong, Nathaniel K C / Gu, Haogao / Ng, Daisy Y M / Chang, Lydia D J / Krishnan, Pavithra / Cheng, Samuel S M / Peiris, Malik / Poon, Leo L M

    Influenza and other respiratory viruses

    2022  Band 17, Heft 1, Seite(n) e13084

    Abstract: ... CoV-2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays ... the multiplex RT-ddPCR assays. These assays have a broad range of linearity and good intra-/inter-assay ... the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 and other respiratory ...

    Abstract Background: Measures for mitigation of Coronavirus Disease 2019 (COVID-19) were set to reduce the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 and other respiratory viruses share similar transmission routes and some common clinical manifestations. Co-circulation of SARS-CoV-2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays for detecting these respiratory viruses is essential for being prepared for future outbreaks of respiratory viruses.
    Methods: A panel of three reverse transcription droplet digital PCR (RT-ddPCR) assays were developed to detect 15 different human respiratory viruses. Evaluations of its performance were demonstrated. A total of 100 local and 98 imported COVID-19 cases in Hong Kong were screened for co-infection with other common respiratory viruses.
    Results: All detected viral targets showed distinct signal clusters using the multiplex RT-ddPCR assays. These assays have a broad range of linearity and good intra-/inter-assay reproducibility for each target. The lower limits of quantification for all targets were ≤46 copies per reaction. Six imported cases of COVID-19 were found to be co-infected with other respiratory viruses, whereas no local case of co-infection was observed.
    Conclusions: The multiplex RT-ddPCR assays were demonstrated to be useful for screening of respiratory virus co-infections. The strict preventive measures applied in Hong Kong may be effective in limiting the circulation of other human respiratory viruses. The multiplex assays developed in this study can achieve a robust detection method for clinical and research purposes.
    Mesh-Begriff(e) Humans ; SARS-CoV-2 ; COVID-19/diagnosis ; Reverse Transcription ; Coinfection/diagnosis ; Coinfection/epidemiology ; Reproducibility of Results ; Real-Time Polymerase Chain Reaction/methods
    Sprache Englisch
    Erscheinungsdatum 2022-12-14
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2274538-5
    ISSN 1750-2659 ; 1750-2640
    ISSN (online) 1750-2659
    ISSN 1750-2640
    DOI 10.1111/irv.13084
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