IN PARTNERSHIP
RNA extraction methods showing very contrasting performances; the transport buffers also displayed clear interactions between kit and buffer. The best and worst kit/buffer combination varied more than five PCR cycles, equivalent to a 30-fold difference in RNA detection sensitivity.
Quality control The platform is approved by the Belgian Federal Institute for Public Health (Sciensano), the Belgian Federal Agency for Medicines and Health Products (FAMHP) and the Belgian National Reference Laboratory. To safeguard quality, Biogazelle has multiple controls in each experiment, namely an internal spike in RNA that controls the RNA extraction and RT-qPCR of each sample, and two positive and negative workflow controls per batch of 99 samples. It has also introduced digital PCR as an orthogonal validation method to calibrate the platform.
Sensitivity of the platform and limit of detection The platform was able to correctly identify five out of five positives and three out of three negatives in an external quality assessment. Of note, from the almost 500 labs that participated, 15% could not detect SARS-CoV-2 RNA in the low-positive sample with 200 copies/ml – equivalent to 20 copies into the RNA extraction or four copies into the qPCR. Also, almost 1% of the labs reported false positives in the no-virus control and 2–3% had false-positive results when testing other strains of the virus. The limit of detection (LOD) is defined as the
lowest quantity at which >95% of replicates are detected. To test the LOD, Biogazelle dispensed 32 replicates of six, three, one and zero cDNA copies (digital PCR calibrated copies). It did not detect any signal in the negative controls, and detected all replicates with six copies per reaction. When using three copies, it lost signal in five wells. This means that the limit of detection is between three and six copies per reaction.
A future-proof test through constant innovation Despite its slow mutation rate, SARS-CoV-2 accumulates mutations as it persists in the human population. It is important to evaluate PCR-based diagnostic assays as new SARS- CoV-2 genome sequences become available. Based on the Covid-19 Genome Analytics in Edge Bioinformatics from Los Alamos National
Laboratory, US, 99.5% of more than 50,000 SARS- CoV-2 genomes are detectable with the E gene assay, and 98.03% with the N gene. Using the two assays at the same time does not only slightly increase analytical detection sensitivity, but also strains coverage to 99.99%. Moreover, the Belgian institute for public health
Sciensano estimates that, in early 2021, Belgium will face 40,000 daily consults of flu-like symptoms for a period of 100 days. Most of these will – hopefully – be free of SARS-CoV-2 but, when doing a test to rule out Covid-19, it would prove valuable to determine if the patient is infected by influenza or respiratory syncytial virus (RSV). Biogazelle is, therefore, developing a seven-target, four-colour multiplex assay to co-detect SARS-CoV-2, influenza A and B, and RSV A and B. The custom-testing platform is scalable, low-cost and has already been used in a diagnostic setting to process hundreds of thousands of patient samples. To its industrial partners, Biogazelle offers:
GCLP-compliant, high-throughput diagnostic testing services for SARS-CoV-2 in an ISO17025 lab environment via RT-qPCR
absolute quantification of a viral load through digital PCR
validation and optimisation of custom SARS- CoV-2 PCR assays
high-throughput RNA expression profiling of therapeutic candidates to elucidate effects on cellular pathways and provide insights on potential toxicity
RNA biomarker analysis in clinical trial samples, to predict or monitor patient response.
Through the offers available, Biogazelle’s experts can help its partners with their Covid-19 vaccine or drug pipeline. ●
https://services.biogazelle.com Outsourcing in Clinical Trials Handbook | 93
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120
