Comparison of the diagnostic efficacy between two PCR test kits for SARS‐CoV‐2 nucleic acid detection

Abstract Background To compare the diagnostic efficacy between two different real‐time reverse transcription polymerase chain reaction (RT‐PCR) test kits for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleic acid detection and provide references for laboratories. Methods Throat swab samples from 18 hospitalized patients were clinically diagnosed with coronavirus disease 2019 (COVID‐19) and 100 hospitalized patients without COVID‐19 were collected. SARS‐CoV‐2 nucleic acid was detected in throat swab samples with RT‐PCR test kits from Sansure Biotech Inc (Hunan, China) and Shanghai BioGerm Medical Biotechnology Co., Ltd.(Shanghai, China). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa value were analyzed, and three parallel tests were performed with three weakly positive samples. Results The sensitivity, specificity, PPV, NPV, and kappa value of the Sansure PCR kit were 0.833, 1.000, 1.000, 0.971, and 0.894, respectively, and the sensitivity, specificity, PPV, NPV, and kappa value of the BioGerm PCR kit were 0.944, 1.000, 1.000, 0.990, and 0.966, respectively. For the three parallel tests, the coefficient of variation value of the BioGerm PCR kit in all three samples was the smallest for both the ORF1ab and N gene. Conclusion The detection efficacy of the BioGerm PCR kit for SARS‐CoV‐2 nucleic acid detection was relatively higher than that of the Sansure PCR kit.


| INTRODUC TI ON
In the final months of 2019, a cluster of pneumonia cases of unclear etiology was first noted in Wuhan, Hubei, China. The etiology of these pneumonia cases was soon identified as a new type of coronavirus. 1 This virus was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the disease it causes is known as coronavirus disease 2019 (COVID-19). 2 Thus far, more than 84,000 COVID-19 cases have been identified in China. Globally, approximately 5.2 million cases were reported as of May 23, 2020 (https://gisan ddata.maps.arcgis.com/apps/opsda shboa rd/index. html#/bda75 94740 fd402 99423 467b4 8e9ecf6). The pandemic has created an enormous burden on health systems, as well as society and the global economy. Successful management of the spread of COVID-19 depends on the timely and accurate diagnosis of patients with acute SARS-CoV-2 infection and accurate detection of asymptomatic carriers. However, the common clinical symptoms and laboratory examination findings of COVID-19 are not unique. 3 Real-time reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive and specific assay and therefore has become the current standard diagnostic method for the diagnosis of COVID-19. 4,5 According to Zhang et al, 6 specimens such as nasal or throat swabs, sputum, lower respiratory tract secretions, peripheral blood, and feces from patients with COVID-19 can be used to detect SARS-CoV-2 nucleic acid. A positive SARS-CoV-2 nucleic acid result from respiratory tract or blood samples is the basis of clinical diagnosis, and two consecutive negative nucleic acid test results are one of the standards for being discharged from the hospital. 7 Thus far, many COVID-19 RT-PCR kits have become commercially available, and the majority of them use the open reading frame 1ab (ORF1ab) and the nucleocapsid protein as the major testing targets. 8 As of May 23, 2020, the Foundation for Innovative New Diagnostics had listed 317 molecular assays on their website as being on the market (www. finddx.org/covid -19/pipeline). However, independent assessment of these products is not yet publicly available. Maximization of the sensitivity and specificity of these test kits is critical to global efforts to control the spread of SARS-CoV-2.
To compare and analyze the detection performance of different SARS-CoV-2 nucleic acid detection kits, two kinds of domestic reagents were selected for parallel detection of a series of samples from Liuzhou People's Hospital in Guangxi, China, which is a designated hospital for patients with COVID-19, to provide references for laboratories.

| Nucleic acid extraction
Nucleic acid was extracted from the samples using magnetic beads following the manufacturer's recommended protocol (Zhongyuan, Chongqing, China). Briefly, throat swab samples from both patients with COVID-19 and patients without COVID-19 were first inactivated with a water bath at 56°C for 30 minutes. Then, 200 μL of the inactivated sample was transferred to a 1.5-mL reaction tube with working buffer (250 μL extraction buffer I + 250 μL extraction buffer II + 4 μL magnetic beads + 15 μL protease K) and heated at 55°C for 4 minutes. Samples were placed in the magnetic bead separator to remove the supernatant before extraction buffer III (600 μL) was added. Afterward, the supernatant was removed again in the magnetic bead separator, and 40 μL eluent was added to separate the extracted nucleic acid from the magnetic beads. Finally, the samples were placed in the magnetic bead separator for 3 minutes to remove the magnetic beads.

| qRT-PCR analysis
A volume of 20 and 5 μL nucleic acid that was extracted from patients with COVID-19, and patients without COVID-19 was subjected to analysis with the previously mentioned Sansure and BioGerm PCR kits, respectively. Amplification was performed using Applied Biosystems ™ 7500 Real-Time PCR system (Thermo Fisher Scientific) with the following protocols: (a) For the Sansure PCR kit, there was an initial 50°C, 30-minutes step for reverse transcription followed by a 95°C, 1-min cDNA pre-denaturation step, then 45 cycles at 95°C for 15 seconds and 60°C for 30 seconds for denaturation, annealing (with fluorescence monitoring), and an elongation step, and finally a 25°C, 10-s step for instrument cool down;  (b) For the BioGerm PCR kit, there was an initial 50°C, 10-minutes step for reverse transcription followed by a 95°C, 5-min cDNA pre-denaturation step, then 40 cycles at 95°C for 10 seconds, and 60°C for 40 seconds for denaturation, annealing (with fluorescence monitoring), and an elongation step. Quality control and assurance, including three internal positive controls and a negative control, were included in each run to identify the false-negative and falsepositive results. Furthermore, three parallel tests were performed with three weakly positive samples, the test for each sample was conducted simultaneously, using two different PCR kits but the same amplification machine.

| Statistical analyses
The  These results indicated that the detection efficacy of the BioGerm PCR kit for SARS-CoV-2 nucleic acid detection was relatively higher than that of the Sansure PCR kit.

| RE SULTS
For the three parallel tests, the results of the Sansure PCR kit showed one of the ORF1ab from sample 2 was not detected. As shown in Table 3, the CV value of the BioGerm PCR kit in all three samples was the smallest for both the ORF1ab and N gene, indicating that the reproducibility of in-batch detections with the BioGerm PCR kit was better than that with the Sansure PCR kit.

| D ISCUSS I ON
Herein, we compared two commercially available RT-PCR kits for the detection of SARS-CoV-2 in clinical samples. These two kits had the same specificity and PPV for SARS-CoV-2 nucleic acid detection; however, the sensitivity, NPV, and kappa value of the BioGerm PCR kit were all higher than those of the Sansure PCR kit; for the parallel tests, the CV value of the BioGerm PCR kit in all three samples was also smaller and more stable than that of the Sansure PCR kit, suggesting that the detection efficacy of the BioGerm PCR kit for SARS-CoV-2 nucleic acid was better than that of the Sansure PCR kit.
COVID-19 is an emergent public health hazard, and its outbreak has caused reagent manufacturers to develop and obtain approval for nucleic acid testing kits in a short time, which may have led to some defects in setting the performance parameters of the kits.
Therefore, in-house clinical validations upon implementation of novel RT-PCR kits need to be conducted. Thus far, several studies have been devoted to this topic, but the majority of them assessed these products using different kits. 9 In summary, we reported the detection performance of two different SARS-CoV-2 nucleic acid detection kits using clinical samples.
While both the assays that were evaluated were highly specific, the BioGerm PCR kit was more sensitive than the Sansure PCR kit. These findings provide important information for the ongoing optimization of viral detection assays following the emergence of COVID-19.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.