key: cord-0878873-p7cxi0q8
authors: Wen, Donghua; Yang, Simin; Li, Guangbo; Xuan, Qiankun; Guo, Wenzheng; Wu, Wenjuan
title: Sample-to-Answer and Routine Real-Time Reverse Transcription PCR (rRT-PCR): A Comparison of Different Platforms for SARS-CoV-2 Detection
date: 2021-03-08
journal: J Mol Diagn
DOI: 10.1016/j.jmoldx.2021.02.010
sha: 9222c8ead9a81d4c25299b82d4463487e06f2217
doc_id: 878873
cord_uid: p7cxi0q8

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading all over the world and has causes millions of deaths. Several Sample-to-Answer platforms, including Cepheid Xpert Xpress SARS-CoV-2 (Xpert Xpress), have received emergency use authorization (EUA) for SARS-CoV-2 nucleic acid detection as a point of care test in the US. But their application niche is unclear comparing with real-time reverse transcription PCR (rRT-PCR) assays cleared by the National Medical Products Administration (NMPA) in China. In this study, the clinical performance, sensitivity and workflow of Xpert Xpress and two rRT-PCR kits (BioGerm, Shanghai and Sansure, Hunan) from 86 symptomatic patients were evaluated. The positive percent agreement (PPA) of Xpert Xpress was 100%, followed by BioGerm Kit (96.15%) and Sansure Kit (90%), respectively. The negative percent agreement (NPA) was 100% for three assays. The LoD is 100 copies/mL for Xpert Xpress, 500 copies/mL for BioGerm Kit and Sansure Kit. By serially diluting five positive specimens assay, the Xpert Xpress displayed better detection capability. In the workflow and throughput analysis, the turnaround time was 51 min for Xpert Xpress, 150 min for BioGerm kit and 210 min for Sansure kit. This study provides some indication for diagnosis methods selection.

The novel coronavirus disease , caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a pandemic all over the world (1) (2) (3) . As of January 31, 2021, more than 101 million infection cases had been confirmed, including more than 2.19 million deaths (https://covid19.who.int/, January 31, 2021).

Until now, SARS-CoV-2, which is a contagion having high rate of transmission from human to human, has been the seventh coronavirus known to infect humans (4) . People contracted with the SARS-CoV-2 can cause mild to severe respiratory illness and symptoms include fever, cough, myalgia or fatigue, sputum production and headache (5) . Additionally, older people, those with underlying medical problems (such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer et al.) are more likely to develop serious illness (https://www.who.int/health-topics/coronavirus#tab=tab_1, 31 January, 2021).

Given SARS-CoV-2 has high rate of transmission and can cause the death of human beings, the foremost priority to facilitate public health intervention and to contain COVID-2019 is reliable laboratory diagnosis. Real-time reverse transcription PCR (rRT-PCR) is one of the routine methods for diagnosis of pathogen in contagion. The detection of pathogen by rRT-PCR was performed in the traditional molecular laboratory by staffs with special technical training. Recently, several sample-to-answer platforms were approved to detect SARS-CoV-2 by the Food and Drug Administration (FDA) in USA, such as Cepheid Xpert Xpress SARS-CoV-2 assay, Abbott ID NOW COVID-19 assay, and GenMark ePlex SARS-CoV-2 assay by FDA (6) . However，there is lack of the comparison between sample-to-answer assay and routine rRT-PCR. Due to the good performance of Cepheid Xpert Xpress SARS-CoV-2 platform (6) , it was compared with two traditional rRT-PCR kits (Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit (PCR-Fluorescence Probing) from Shanghai BioGerm Medical Biotechnology Co.,Ltd, China (BioGerm Kit), and Novel rRT-PCR kits were compared.

Nylon swabs and viral transport tubes with 3 mL preservation medium were prepared. One Pharyngeal Swab and two nasal swabs (pharyngeal-nasal swabs) were collected from each symptomitic patient from fever clinic. The three swabs were placed into a viral transport tube.

Break swabs at the indicated break line and cap the specimen collection tube tightly. Then specimens were transported to the clinic laborary. Before nucleic acids detection, the SARS-CoV-2 was inactivated by 56 o C for 30 min. After routine detection, samples were aliquoted and stored at -80 o C immediately. A number of 34 fresh specimens, including 6 positive and 28 negtive specimens, were detected parallelly by Xpert Xpress and BioGerm Kit.

A total of 86 specimens, including 26 positive specimens and 60 negative specimens, 

For each testing, 300 μL of pharyngeal-nasal swab specimen was added into the sample chamber in the cartridge ultilizing transfer pipette. Then, the lid is closed and the cartridge is 

The SARS-CoV-2 RNA standard substrate (Catalog number: GBW(E)091099) was bought from National Medical Products Administration of China for limit of detection study. The concentration is 6.89×10 5 copies/mL for ORF1ab, 1.36×10 6 copies/mL for N and 8.04×10^5 copies/mL for E .ORF1ab and N were diluted to designated concentrations and detected, respectively. At the designated concentration, five replicates were tested for each gene.

Five specimens with high Ct value (ORF1ab: 18.50-23.56; N: 20.23-24.09 by BioGerm Kit)

were selected for serial dilution using mixture of negative samples. The dilution ratio were1:10 1 , 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 and 1:10 6 , respectively. The diluted samples were aliquoted and tested immediately by Xpert Xpress, BioGerm Kit and Sansure assay in parallel.

Positive rate were calculated and used to evaluate the sensitivity of three testing methods.

Anti (Table 2 ). It was suggested that those two COVID-2019 patients were convalescents.

The limit of detection (LoD) was determined utilizing pure SARS-CoV-2 RNA from National Medical Products Administration of China. Before testing, the SARS-CoV-2 RNA was diluted in negative clinical specimen. The positivity rate observed was ≥100% at a lowest SARS-CoV-2 concentration of 100 copies/mL for Xpert Xpress, 500 copies/mL for BioGerm Kit and Sansure Kit (Table 3) . So, the LoD is 100 copies/mL for Xpert Xpress, 500 copies/mL for BioGerm Kit and Sansure Kit.

In order to evaluated the sensitivity of three methods to the clinic specimens, five positive ability; but when the dilution ratio was more than 1:10 5 , the Xpert Xpress had better detection capability ( Table 4) . 

As the SARS-CoV-2 is a highly infectious virus which can spread by person-to-person (7), Biotechnology has also been granted by Chinese government. The aim of this study was to comparison the comprehensive performance between sample-to-answer platform and routine rRT-PCR kits.

Because of the good performance of Xpert Xpress from Cepheid (6) (Table 2) . By detecting serial dilution samples derived from five positive specimens, when the dilution ratio was more than 1:10^5, the Xpert Xpress had better detection capability. The judgments were concluded strictly according to the instructions of the kits (Table 3 and In conclusion, we compared the analytical and clinical performance and the workflow of FDA-cleared Xpert Xpress SARS-CoV-2 assay platform with two NMPA-cleared routine rRT-PCR kits. Xpert Xpress SARS-CoV-2 assay platform has better clinical performance, sensitivity compared with routine rRT-PCR. In addition, Xpert Xpress SARS-CoV-2 assay platform is more convenient to operate, but it is limited by its low throughput. Xpert Xpress, ample-to-answer molecular diagnostic platform for single specimen detection, can be applied to detect emergency cases. Routine rRT-PCR assays, with batch detection capacity, are suitable for epidemiological screening of large population. This study provides some indication for diagnosis methods selection. J o u r n a l P r e -p r o o f 

A novel coronavirus outbreak of global health concern

A Novel Coronavirus from Patients with Pneumonia in China

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Laboratory diagnosis of emerging human coronavirus infections -the state of the art

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet

Clinical Evaluation of Three Sample-To-Answer Platforms for the Detection of SARS-CoV-2

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Active microelectronic array system for DNA hybridization, genotyping and pharmacogenomic applications

We thank Cepheid for providing the cartridges via an Investigator-Initiated Study agreement (Cepheid-IIS-2020-00xx) and Sansure for providing the nucleic acid purification and detection reagent.