Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses
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Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses. / Tang, Hui Ting; Nörz, Dominik; Grunwald, Moritz; Giersch, Katja; Pfefferle, Susanne; Fischer, Nicole; Aepfelbacher, Martin; Rohde, Holger; Lütgehetmann, Marc.
In: J CLIN VIROL, Vol. 173, 08.2024, p. 105693.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses
AU - Tang, Hui Ting
AU - Nörz, Dominik
AU - Grunwald, Moritz
AU - Giersch, Katja
AU - Pfefferle, Susanne
AU - Fischer, Nicole
AU - Aepfelbacher, Martin
AU - Rohde, Holger
AU - Lütgehetmann, Marc
N1 - Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.
PY - 2024/8
Y1 - 2024/8
N2 - BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %.DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.
AB - BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %.DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.
KW - Humans
KW - Multiplex Polymerase Chain Reaction/methods
KW - Respiratory Tract Infections/virology
KW - Sensitivity and Specificity
KW - High-Throughput Screening Assays/methods
KW - Viruses/isolation & purification
KW - Virus Diseases/diagnosis
KW - Automation, Laboratory/methods
KW - SARS-CoV-2/genetics
KW - COVID-19/diagnosis
KW - Molecular Diagnostic Techniques/methods
U2 - 10.1016/j.jcv.2024.105693
DO - 10.1016/j.jcv.2024.105693
M3 - SCORING: Journal article
C2 - 38820916
VL - 173
SP - 105693
JO - J CLIN VIROL
JF - J CLIN VIROL
SN - 1386-6532
ER -
