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. 2021 Mar:289:114042.
doi: 10.1016/j.jviromet.2020.114042. Epub 2020 Dec 17.

Evaluation of extraction and amplification assays for the detection of SARS-CoV-2 at Auckland Hospital laboratory during the COVID-19 outbreak in New Zealand

Indira Basu  1 , Radhika Nagappan  2 , Shivani Fox-Lewis  3 , Sharmini Muttaiyah  4 , Gary McAuliffe  5
Affiliations

Evaluation of extraction and amplification assays for the detection of SARS-CoV-2 at Auckland Hospital laboratory during the COVID-19 outbreak in New Zealand

Indira Basu et al. J Virol Methods. 2021 Mar.

Abstract

Utilising diverse molecular platforms has formed a solid foundation in New Zealand's COVID-19 response. We evaluated multiple extraction and PCR assays for the detection of SARS-CoV-2. We included 65 positive samples which were run on the Panther Fusion using a laboratory developed test (LDT, E gene target). Where viral RNA was extracted by MagNA Pure (MP) 96 extraction platform or EpMotion 5075/Geneaid extraction kit, SARS-CoV-2 detection was performed on Light Cycler (LC) 480 using a LDT (E gene) or 3 commercial assays; Certest Viasure (Orf1ab, N genes) GenePro (E, RdRp genes) and A* Star Fortitude (proprietary target). Median Cts on LC 480 LDT for specimens (n = 9) extracted on MP 96 (26.6) were lower than on EpMotion (31.6) whereas median Cts for specimens (n = 10) extracted on the Panther Fusion LDT (23.1) were comparable with MP 96 /LC480 LDT (23.6). Specimens tested on Panther Fusion LDT (n = 28), extracted by MP 96, and amplified using commercial assays showed good concordance with a few exceptions; lower median Ct values were seen for 2 targets on GenePro (16.9, 21.5) and Viasure (19.5, 21.1) than for the Panther Fusion LDT (24.2) and A* Star Fortitude (25.6). Specimens tested on MP 96 (n = 18) had comparable results using commercial assays, with lower median Cts for Viasure (22.2, 23.7) compared with the LC 480 LDT (24.7), GenePro (24.7,25.7) and A*Fortitude (25.1) assays. The study provides an early assessment of the performance characteristics of 3 extraction methods for viral RNA and 5 PCR assays for the detection of SARS-CoV-2.

Keywords: COVID-19; New Zealand; RT-PCR; SARS-CoV-2; Viral RNA extraction.

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Conflict of interest statement

The authors report no declarations of interest.

Figures

Fig. 1
Fig. 1
below shows box plots comparing the cycle threshold, Ct using the LDT Light Cycler 480 [n = 9] when specimens were extracted using MagNAPure 96 extraction versus when specimens were extracted using EpMotion 5075/Geneaid Viral RNA extraction kit. Nine specimens extracted on MP 96 extraction with a median Ct of 26.6 (interquartile range, IQR 23.3-28) had a higher median Ct of 31.6 (IQR 29.1–33.8) when extracted on the EpMotion 5075/Geneaid Viral RNA extraction kit and amplified using the LDT on the LC 480.
Fig. 2
Fig. 2
below shows box plots comparing the cycle threshold, Ct using Panther Fusion LDT (extraction and amplification) versus MagNAPure 96 extraction/Light Cycler 480 LDT [n = 10]. Three out of the 10 specimens were tested on the EpMotion were stored at 4 °C for longer than 1 week. Ten specimens processed on the MP 96/LC 480 using the LDT with a median Ct of 23.6 (IQR 17.5–27.6) had comparable results on the Panther Fusion LDT with a median Ct of 23.1 (IQR 18.4–27.9) for the same samples.
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