key: cord-0871712-c8i4gyvc
authors: Ye, Qing; Lu, Dezhao; Zhang, Ting; Mao, Jianhua; Shang, Shiqiang
title: Application experience of a rapid nucleic acid detection system for COVID-19
date: 2022-01-31
journal: Microbes Infect
DOI: 10.1016/j.micinf.2022.104945
sha: fbd90e425d8a0a3d7e23cea0886d761db08b1a65
doc_id: 871712
cord_uid: c8i4gyvc

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging worldwide. The COVID-19 outbreak caused severe threats to the life and health of all humans caused by SARS-CoV-2. Clinically, there is an urgent need for an in vitro diagnostic product to detect SARS-CoV-2 nucleic acid quickly. Under this background, commercial SARS-CoV-2 nucleic acid POCT products came into being. However, how to choose these products and how to use these products in a standardized way have brought new puzzles to clinical laboratories. This paper focuses on evaluating the performance of these commercial SARS-CoV-2 nucleic acid POCT products and helps the laboratory make the correct choice. At the same time, to standardize the use of this kind of product, this paper also puts forward corresponding suggestions from six elements of total quality management, namely, human, machine, material, method, environment, and measurement. In addition, this paper also puts forward some ideas on the future development direction of POCT products.

detection and automatically complete detection and result analysis [ 6] .

POCT has many advantages, but it also brings some new problems to the clinical laboratory. For example, whether a gene amplification testing laboratory is needed, a professional operation is required, operator biosafety protection level, etc. This paper focuses on the commercial products of nucleic acid detection POCT in SARS-CoV-2 and the requirements of the detection system. In addition, the future development direction.

To contain and help stop the spread of COVID-19, rapid, precise, and large-scale detection of SARS-CoV-2 is crucial. The need for a sensitive, user-friendly, and rapid diagnostic test has become increasingly urgent. Currently, many commercial POCT products for SARS-CoV-2 have become available to fulfill this demand. The integration of nucleic acid extraction and amplification and detection processes is usually realized by developing supporting equipment for commercial SARS-CoV-2 nucleic acid POCT products. Putting all the reagents in one box and automating them using mechanical manipulation or microfluidic technology minimizes manual manipulation, reduces the total test time, and improves the turnaround time ( Figure 1 ).

Currently, many commercial SARS-CoV-2 nucleic acid POCT products for COVID- 19 (Seegene Inc.) [7] . Studies on the performance of these seven FDA-approved and commercially available SARS-CoV-2 nucleic acid POCT products showed that the overall performance of commercial SARS-CoV-2 nucleic acid POCT products was high, with a summary sensitivity of 95.9% (95% CI 93.9-97.2%, I2 = 60.22%) and specificity of 97.2% (95% CI 95.5-98.3%, I2 = 56.66%) [8] [9] [10] [11] [12] [13] [14] . However, the ID NOW COVID-J o u r n a l P r e -p r o o f 19 (Abbott) and the Simplex COVID-19 Direct exhibited lower sensitivity than other platforms, consistent with previously reported studies [9] [10] [11] [12] [13] [14] .

Among these products, Xpert Xpress (∼45 min), ID Now Covid-19 (∼15 min), and Biofire COVID-19 test (∼50 min) can complete the detection within 1 hour. Xpert Xpress SARS-CoV-2 detects the E gene and N2 gene by RT-PCR and is performed on the GenExpert instrument system [15, 16] . Briefly, 300 μl of the sample was added to the testing tube and mixed upside down five times when testing. Then, the tube was tested on an instrument, and it took approximately 45 minutes to complete the test. ID NOW is a rapid molecular in vitro diagnostic reagent for detecting the RNA-dependent nucleic acid rapid detection system adopts the independent temperature control design of a single sample hole, which realizes the follow-up detection of samples and provides a more convenient scheme for improving the efficiency of nucleic acid detection in emergencies. Many actual clinical samples have verified the system; its sensitivity and specificity are over 95%, and the lowest detection limit can reach 400 copies/mL. DX-2A adopts microfluidic integrated nucleic acid extraction and purification technology and nested isothermal amplification technology to realize high-sensitivity amplification, detect trace viral nucleic acids, and complete full-closed rapid detection within 45 minutes.

The current COVID-19 pandemic demands massive testing-based nucleic acid assays, which are considered the gold standard diagnostic test for detecting the SARS-CoV-2 virus. However, mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits lead to false-negative results [17] [19] . For many of the systems, hardly any real-world evidence is available yet.

While next-generation sequencing (NGS) is recognized as the gold standard for SARS-CoV-2 variant identification and characterization of mutations in the viral genome [20] , however, it is expensive, so there is no way to use it for routine testing.

This multiplex real-time RT-PCR, which identifies certain SNPs specific to VOCs, appears to be a fast, cheaper and less technically demanding method to generate data regarding the spread of different SARS-CoV-2 variants and is a suitable method for J o u r n a l P r e -p r o o f lower-income countries to supplement the data generated by genomic sequencing [20] .

The six factors of "human, machine, material, method, environment and measurement" are the main factors in the total quality management theory, referred to as "six elements" management for short. POCT of SARS-CoV-2 nucleic acids also need to follow this theory ( Figure 2 ).

Although the POCT platform integrating nucleic acid extraction and amplification detection in COVID-19 is easy to operate, the whole experimental process still involves many aspects, such as preventing pollution, performance verification, equipment maintenance, indoor quality control, result analysis and reporting, and laboratory biosafety. Therefore, POCT detection laboratory personnel still need to pass the prejob training. Practitioners should obtain corresponding qualifications and pass SARS-CoV-2 nucleic acid detection-related training and workability assessment before they can take up the post.

At the present stage, the opening of the sampling tube and the loading of samples of instant nucleic acid detection products in SARS-CoV-2 have not been automated.

Therefore, the laboratory should be equipped with enough instruments and equipment needed for the POCT of SARS-CoV-2 nucleic acids, including the biosafety cabinet.

Other necessary equipment includes refrigerators for storing reagents and specimens, sample applicators, necessary virus inactivation equipment, centrifuges, portable ultraviolet lamps, nucleic acid instant detection equipment, and uninterruptible or standby power supply. The laboratory should also establish procedures for the use, maintenance, and calibration of nucleic acid instant detection equipment and carry out maintenance and regular calibration according to the procedures in daily work to ensure the normal operation of these instruments and equipment.

J o u r n a l P r e -p r o o f

The laboratory should use highly sensitive (detection limit ≤500 copies/ml) detection reagents approved by the National Medical Products Administration. POCT reagents must provide the analytical performance of the whole POCT system, including sample sampling tubes, nucleic acid extraction reagents, and nucleic acid detection reagents. Performance parameters include but are not limited to the detection limit and precision. If the POCT reagent simplifies the nucleic acid extraction process, such as using a one-step method, the extraction efficiency and purity of nucleic acids will be affected by the components of the sample preservation solution in the sample sampling tube, thus affecting the performance of POCT [21] . Therefore, the laboratory should choose the sample sampling tube recommended by the POCT reagent manufacturer as much as possible. If other sampling tubes are used, the performance must be confirmed first, and the detection performance of the system cannot be used until it is evaluated.

The laboratory should establish standard operating procedures for the whole process of detection operation according to the reagent instructions. Before clinical testing, the laboratory should verify the whole POCT system (including recommended sample sampling tubes, nucleic acid extraction reagents, and nucleic acid detection reagents). For performance verification, known concentrations of pseudovirus-positive quality control products packaged with corresponding viral RNA sequences and negative clinical specimens can be used. Performance indicators include but are not limited to the detection limit and precision [4] . Precision should include the repeatability of different test batches of the same instrument and the same operator, reproducibility between different operators of the same instrument, and reproducibility of the same operator using different batches of reagents on different instruments. The laboratory should also compare the difference in detection limit between POCT reagents and conventional nucleic acid detection reagents being used in the laboratory.

Gradient dilution of positive quality control products was carried out using the preservation solution in the sample sampling tube recommended by the two reagents, and each gradient was repeated no less than five times for nucleic acid extraction and J o u r n a l P r e -p r o o f detection. The detection limit is the lowest detectable concentration. If the laboratory is equipped with multiple POCT instruments, the laboratory should also evaluate the reproducibility between different instruments.

The laboratory should carry out internal quality control. Internal quality control products in the laboratory shall include negative quality control products (normal saline)

and weak positive quality control products (third-party quality control products, concentration can be 1.5~3 times of the detection limit). Weak positive internal quality control products and negative internal quality control products should be tested before testing clinical specimens. Only after internal quality control in the laboratory is qualified can clinical specimens be tested. Generally, when startup detection reaches 24 hours or less than 24 hours, but the number of continuously detected samples reaches a certain amount, weak positive internal quality control products should be tested again to monitor whether the POCT system is still under control. In addition, it is necessary to participate in the external quality assessment to verify the accuracy.

The laboratory should analyze and explain the results according to the reagent instructions. Comprehensive judgment should be combined with the original amplification curve for POCT equipment that can automatically report the results and display the original amplification curve. When the detection limit of the instant detection system is ≤500 copies/ml, the detection result is negative, and the negative result can be directly reported. However, when the test result is positive, 1~2 other kinds of more sensitive conventional nucleic acid detection reagents for amplifying different target regions should be used for verification. If necessary, the patient can be resampled for re-examination, and the results can only be reported if the re-examination is positive.

In principle, the PCR laboratory should set up reagent storage and preparation areas, specimen preparation areas, amplification, and product analysis areas. For the POCT detection platform integrating nucleic acid extraction and amplification detection, the specimen preparation area and amplification and product analysis area J o u r n a l P r e -p r o o f can be combined. However, opening the lid and adding samples in an A2 type II biosafety cabinet is necessary.

However, if the detection process needs to open the lid many times and the daily test sample quantity is large, three partitions should still be set up. In addition, the laboratory needs to maintain good ventilation, and it is recommended that the ventilation in all areas of the laboratory be > 12 times/h [22] .

At this stage, both commercial SARS-CoV-2 nucleic acid POCT products need to open the cover of the specimen sampling tube and then add samples. Therefore, the instant detection of SARS-CoV-2 nucleic acids should be carried out in a second-level biosafety laboratory. In addition, the laboratory should make corresponding biosafety risk assessments according to the number of opening covers, whether the virus is inactivated before testing, and the complexity of the detection process of different POCT platforms. On this basis, appropriate personal protective measures should be taken, including gloves, masks, and isolation gowns [4] .

In addition to providing strong support for rapid control of the spread of COVID-19, these research results of instant detection in SARS-CoV-2 can also be applied to emergency defense and rapid deployment of other diseases, which will help to cope with possible emerging infectious diseases in the future. Based on this application scenario, future POCT products need to be characterized by miniaturization, rapidity, simple operation, intuitive results, and biosafety-friendliness [23] .

Nanotechnology is a multidisciplinary field that includes designing, producing, and applying materials at the nanometer scale. Nanomaterials have gained worldwide attention in diagnostics [24] . Their small size and large surface area enhance their surface reactivity, quantum confinement effects, electrical conductivity, and magnetic properties, which have made them potential tools in developing innovative diagnostic systems, including SARS-CoV-2 [25] [26] [27] [28] .

Recently, a CRISPR/Cas-based rapid detection assay has been developed for on-J o u r n a l P r e -p r o o f site COVID-19 diagnosis. CRISPR/Cas is a gene-editing toolbox, a combination of guide RNA (CRISPR RNA or crRNA) and the Cas enzyme complex [29, 30] .

DETECTR are two promising CRISPR-based SARS-CoV-2 detection techniques.

SHERLOCK detected the 2019-nCoV-specific S-and Orf1ab-genes in <1 h with a LOD of 10-100 copies/μl [31] . Using magnetic beads for purification in this method lowered the detection time to 15-45 min with 93.1% sensitivity and 98.5% specificity [32] .

Similarly, a CRISPR-based DETECTR assay for SARS-CoV-2 detection with a 95%

positive prediction agreement within 30 minutes was developed [33] . Additionally, a paper strip-test based on the CRISPR-Cas9 analytical tool FNCAS9 Editor-Linked

Uniform Detection Assay (Feluda) was developed, exhibiting a sensitive tool toward SARS-CoV-2 detection that provides results within minutes.

All these technologies are the way forward.

The POCT method has some advantages over the conventional RT-PCR method.

However, it still cannot replace the traditional RT-PCR method at the present stage because the detection sensitivity and specificity of these POCT methods have not exceeded those of the traditional RT-PCR method. Therefore, we need to control its use scenarios reasonably. Importantly, antibody and nucleic acid tests should complement each other to improve the diagnosis, especially to screen asymptomatic patients better and reduce the false-negative phenomenon of "false recovered patients" or premorbid patients with low virus latency [34] . Since nucleic acid POCT technology integrates nucleic acid extraction, amplification, and detection and automatically completes detection and result analysis, it is more rapid than classic PCR. The principle of RT-PCR is that reverse transcriptase is used to convert RNA into its complementary cDNA, the specific region of the cDNA is amplified by polymerase chain reaction (A); LAMP-PCR first reverse transcribes the RNA genome of SARS-CoV-2 into cDNA, then designs four specially designed primers that can bind to six different regions of the target genome, and uses DNA polymerase with strand displacement activity instead of thermal denaturation to generate a single-stranded template (B); RPA-PCR mainly depends on three enzymes:

recombinase, which can bind single-stranded nucleic acids, single-stranded DNA binding protein (SSB), and strand displacement DNA polymerase, is also an isothermal amplification method of nucleic acids (C); NEAR-PCR is driven by reverse

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