key: cord-0697688-rfw1vvfv
authors: Artik, Yakup; Coşğun, Alp B.; Cesur, Nevra P.; Hızel, Nedret; Uyar, Yavuz; Sur, Haydar; Ayan, Alp
title: Comparison of COVID‐19 laboratory diagnosis by commercial kits: Effectivity of RT‐PCR to the RT‐LAMP
date: 2022-01-21
journal: J Med Virol
DOI: 10.1002/jmv.27559
sha: 9e772d2cc36d3ece45ca171d35e5c391c85ac8ee
doc_id: 697688
cord_uid: rfw1vvfv

Coronavirus disease 2019 or COVID‐19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 or 2019‐nCoV) is an ongoing pandemic that has emerging global effects and requires rapid and reliable diagnostic testing. Quantitative reverse transcription‐polymerase chain reaction (q‐RT‐PCR) is the gold standard method for SARS‐CoV‐2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as one of these novel approaches may also contribute to faster and cheaper field‐based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30–45 min and to compare the effectiveness of LAMP to the q‐RT‐PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS‐CoV‐2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT‐LAMP as well as by conventional q‐RT‐PCR. The patient samples were tested with four different kits (SENSObiz COVID‐19 [SARS‐CoV‐2] LAMP Assay, the QIAseq DIRECT SARS‐CoV‐2 kit, Biospeedy SARS‐CoV‐2 Variant Plus kit, and CoVirion‐CV19‐2 SARS‐CoV‐2 OneStep RT‐PCR kit) and two different PCR devices (GDS Rotor‐Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor‐Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor‐Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS‐CoV‐2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.

depending on their viral loads that represent the ratio range from 8% to 80%. 3 Incubation time is significant to detect the disease clinically and it was updated as 6.4 days with recent studies. 4 The coronavirus family members are positive-sense enveloped single-stranded RNA viruses that are categorized into four genera as beta, alpha, gamma, and delta coronavirus. The SARS-CoV-2 is a beta type and only affects mammals. Mainly, it is composed of a 30 kb genome with 14 open reading frames (ORFs) encoded to the spike protein (S), nucleocapsid protein (N), a small membrane protein (SM), and membrane glycoprotein (M) with an additional membrane glycoprotein (HE). 5 The spike protein is the actual part for binding the specific host receptor, angiotensin-converting enzyme 2 (ACE2).

When the spike protein binds to the receptor, it divides two main subunits; first, an amino-terminal subunit (S1) and a carboxyl-terminal subunit (S2) by host furin-like proteases, as shown in Figure 1 . 6 The genetic sequence of SARS-CoV-2 shares 79.5% with SARS-CoV and 96.2% identical genome with RaTG13 which is a short RNAdependent RNA polymerase (RdRp) region that originated from bats of CoV. 7 The RT-PCR is a quantitative and gold standard method that is commonly preferred. In this technique, three main genes are targeted for SARS-CoV-2 virus detection including the N-gene (N protein), Orf1b gene (human RNA polymerase protein), and the E-gene (E protein). In the RT-PCR technique, the specificity of the confirmatory test relies on the probe-target sequence. 11 patients. On the other hand, the immunoassays are alternatively utilized test types used the enzyme-linked immunosorbent assay (ELISA) that are cheaper but also less sensitive compared to PCR. The immunofluorescence assay (IFA) is utilized for the diagnosis of SARS-CoV-2 with the presence of immunoglobulin G (IgG). 12 The bloodbased tests are serological tests that are used to identify whether the person had an infection. The antibodies are utilized to detect the disease. Antibodies (IgM and IgG) are specific to an antigen within the blood. Principally, the immune system recognizes these antigens of an infected person as a foreign element and specific antibodies can be created to fight the infection within the body. Thus, these antibodies can act as labels for the disease that are generally produced after the second week of the virus infection. Although IgM antibodies can be detected after 10-20 days, IgG is determined after 20 days of SARS-CoV-2 infection. 13 RT-LAMP technique is a recently preferred technique which is a rapid and sensitive method used in SARS-CoV-2 detection. Nucleic acid detections occur over 1 h and this easily interpretable colorimetric assay requires only a heat source. 15 LAMP technique is the fast and low-cost simple colorimetric technique that makes RT-LAMP an effective solution for ramping up global testing capacity. Moreover, it is single tube technology to detect the target nucleic acid sequences. 16 On the basis of the LAMP technique, six primers are utilized, including four primers selected by combining parts of the target DNA and two additional loop primers that are used to amplify a specific gene region. Recently, RT-LAMP has been applied for POC for many RNA virus infections. 17 According to this perspective, LAMP has taken an important place for the diagnosis of virus infections, such as SARS-CoV-2. Thus, in this study, it is aimed to compare the COVID-19 diagnosis effectivity of RT-PCR to the RT-LAMP. In addition, our results contribute to the proactive approach for the development of a rapid diagnosis system. 

In this kit, 6-carboxy-fluorescein (FAM), phosphoramidite (Hex), 6-carbocyl-X-Rhoddamine (ROX) and, carboxylic acid (Cy5) channels were utilized for ORF1ab, RNaseP, Spike (S) gene and, Nucleocapsid (N), respectively. Based on the kit protocol, 2.5 µl patient samples with VTM were added to a 7.5 µl ready kit mixture to achieve a 10 µl PCR mixture in total. Thermal cycle parameters of RT-PCR amplification were as follows: 52°C for 3 min for reverse transcription, 95°C for 10 s for holding, then 35 cycles of 85°C for 1 s and 60°C for 1 s for denaturation, annealing, and extension, respectively.

In this kit, FAM, HEX, and Texas Red were used for S, Orf1ab, and RNaseP gene, respectively. According to the kit protocol, 5 µl patient samples with VTM were added to a 15 µl ready kit mixture to achieve 20 µl PCR mixture in total. Thermal cycle parameters of RT-PCR amplification were as follows; 55°C for 10 min for reverse transcription, 95°C for 10 s for holding, then 40 cycles, and 60°C for 30 s for denaturation, annealing, and extension, respectively.

In this kit, FAM, HEX, and Cy5 were used for S, Orf1ab, and RNaseP gene, respectively. According to the kit protocol, 5 µl patient samples with VTM were added to a 15 µl ready kit mixture to achieve a 20 µl PCR mixture in total. Thermal cycle parameters of RT-PCR amplification were as follows; 50°C for 10 min for reverse transcription, 95°C for 2 min for holding, then 40 cycles, and 58°C for 30 s for denaturation, annealing, and extension, respectively.

In the LAMP procedure, the kit does not require any extra RNA extraction step due to the use of VTM solution, and another advantage is that swap samples can be used just after being taken from the patient. Thus, 

The special rotary design of the Rotor-Gene Q makes it the most precise and versatile real-time PCR cycler currently available. Each tube spins in a chamber and keeps all samples at precisely the same temperature during rapid thermal cycling. Cq value was arranged automatically in Rotor-Gene Table 1 .

The data were analyzed using the SPSS 25.0 package program. The distribution of the data was examined with the Kolmogorov-Smirnov test. Student's t test, one-way analysis of variance (ANOVA), χ 2 , and Fisher Exact test were used for parametric data as well as descriptive statistical methods (mean, standard deviation, frequency) while evaluating the study data. It was calculated in the 95% confidence interval when evaluating the study. Table 2 .

When the kit sensitivity was considered in terms of Cq values, in the first day Rotor-Gene group, no significant difference was found between the Qiagen and Covirion pairs. There was a significant difference between all the other pairs. The Biospeedy kit worked better on the first and fifth days in both the Inovia and the Rotor-Gene device than Covirion and Qiagen. On the other hand, it was concluded that the Covirion kit worked better than the Qiagen kit in both the first and fifth days in the Inovia and Rotor-Gene devices.

When the kit was considered in terms of the positive and negative values of the samples in the Inovia device, there was no difference between the Biospedy and Covirion kit on the first day. The

Qiagen kit was also in the 93.3% confidence interval compared to the Biospedy and Covirion. This confidence interval was insignificant in terms of kit sensitivity and does not reveal any significance, as shown in Table 3 .

When the kit was considered in terms of the positive and negative values of the samples in the Inovia device; the rates of positive and negative groups showed significant differences according to kits in subgroups. There was no difference between the Biospeedy and Covirion kit, and it is observed that the sensitivity of the Qiagen kit was decreased compared to the Biospeedy and Covirion in terms of significance, as summarized in Tables 4 and 5 .

When the Rotor-Gene device was considered the use of the kit in terms of the positivity and negativity values of the samples, there was no difference between the Biospedy and Covrion kit on the first day. Moreover, the Qiagen kit was also in the 63.3% confidence interval compared to the Biospedy and Covirion in terms of significance. This confidence interval was the kit sensitivity, as summarized in Table 6 .

The positivity and negativity values of the samples in the Rotor-Gene device exhibit significant differences according to kits in subgroups. There was no difference between the Biospedy and the Covrion kit. It was observed that the sensitivity of the Qiagen kit was decreased compared to the Biospedy and Covirion kit in terms of significance, as summarized in Table 7 .

When the LAMP group was compared with the inovia and the Rotor-Gene device on the first day, a significance of 63.3% was detected, and when the cost, speed, and laboratory requirement used in rapid diagnosis systems were considered, these results are found to be in an ideal confidence interval as summarized in Tables 7 and 8 .

On the fifth day, when the LAMP group was compared with the Rotor-Gene device and the Inovia device, a 60% significance was Threshold values of the first day were found to be significantly different between the positive and negative groups, as shown in Table 15 , but there was no significant difference between these groups in the Cq value of the fifth day as shown in Table 16 .

Nowadays, SARS-CoV-2 is also defined as a chemical, biological, The problem of RT-PCR with inaccurate results was increasingly exposed. 22 Moreover, RT-PCR requires trained medical staff, specialized instrumentation, technical labor, and special chemicals or reagents. 23 The accuracy of the serological tests is also not enough to detect the SARS-CoV-2 infection. Generally, these tests can be coupled with the RT-PCR based on the presence of viral RNA. 24 The LAMP technique is an alternative to the conventional quantitative RT-PCR methods that do not require expensive instruments to perform the reaction or interpret the results and LAMP may achieve higher sensitivity on crude clinical samples than RT-PCR. 25 RT-LAMP is a nucleic acid amplification assay like RT-PCR, which is a simple, low cost and fast method. 26 Therefore, this is the most important advantage of the RT-LAMP method that distinguishes it from other methods.

The incubation period of the disease is thought to extend to 14 days, with a median time of 4-5 days from exposure to symptoms. 29 A study reported that 97.5% of people with COVID-19 who have symptoms will do so within 11.5 days of SARS-CoV-2 infection. 30 Experiments were carried out on the first and fifth days since the incubation period was 4-5 days for the first occurrence. According to the results (as shown in Table 1 ) positivity was caught ideally by inovia and Rotor-Gene devices for all kits on the first day. However, on the fifth day, the Qiagen kit showed a significant decrease in positivity rate based on the first day, which is 53.3% for the inovia device and 63.3% for Rotor-Gene. On the other side, when the LAMP is examined, the first-day positivity rate was 63.3% and the fifth day was 60%. All experiments were performed on 30 different patients with known positivity. This study focused on the sensitivity of the LAMP-PCR system, whose confidence interval has proven to be above acceptability in urgent needs for pandemic guidelines. It works without the need for any complicated device, especially with its portable device form. At the same time, the denaturation, annealing, and extension steps applied in RT-PCR are not found in the LAMP technique, and the most striking feature is that the whole experiment is performed at constant temperature (54°C). In this way, it is possible to perform these steps even by using only a water bath. As the use of LAMP PCR in this field is very new, comprehensive studies are needed. 31 Also Chaouch et al. revealed that the RT-LAMP method has reliable application for SARS-CoV-2 diagnosis due to its simple application and low technical requirements, thus presenting a potentially effective test to help us to fight the ongoing COVID-19

pandemic. 32 The hope is that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times. 33 This approach could be used for monitoring exposed individuals or potentially aid with screening efforts in the field and potential ports of entry. 20, 34 During the event, the time for the development of the kits and the devices is further shortened. Ultimately, it is an analysis system that can read both quantitatively and qualitatively.

With this study, we foresee it will be used frequently in the future in cases of epidemics or pandemics in many CBRN agents, as well as in the triage stages of the emergency services of hospitals.

Furthermore, the study showed that there may be differences between the kits used in the diagnosis and their results. The results of this study will shed light on further studies to be conducted in larger samples. We also thank Zeynal Abidin Kocadağ for his statistical methodology interpretations. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

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The authors declare that there are no conflict of interests.

The research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. This study protocol was