Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in well plates in per run. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Huseyin Tombuloglu received the awards. Competing interests: The authors have declared that no competing interests exist. The increasing number of infections worldwide necessitates the need for a less-invasive, reliable, and fast diagnostic tool [ 1 ]. To achieve this goal, several studies have tackled this challenge and these efforts have yielded several diagnostic kits. Each of these methods has its own strong and weak points in terms of sensitivity and specificity.
In addition to these strategies, efforts to develop SARS-CoV-2 detection methods with high efficiency and accuracy, with less reaction time and effort, are still ongoing. For the best RT-PCR performance, the combination of these potential gene targets should be optimized.
More than annotated genomes, whose genome sequence information have been determined and which include samples from Europe and Asia, were selected. In this way, virus gene targets are adjusted as sensitive, specific, and accurate as possible. The concentration and sequence of each primer or probe are stated in Table 1. Also, it is approved by the institutional research committee and the de-identified samples were left over after completion of diagnostic tests; hence this study requires no consenting as per institutional ethics committee regulations.
The mixture for the primer and probe is varied according to the kit design. To find out the amplification efficiency E of the genes, a standard curve from the dilution series of templates was prepared. Ct values versus the logarithmic amount of the template were plotted.
The amplification efficiency was obtained by using the following equation: 1. The results were evaluated by determining the amplification curve of the target gene and the internal control gene. For the ABI device, the cycle threshold Ct or Cq line was automatically adjusted to ensure that the curves are all straight position.
For this purpose, ABI software v2. The average cycle threshold Ct value with standard deviations SD were Triplicate reactions yielded the Ct value as In the second reaction mixture, the Ct value was detected as Three primer and probe set for each gene were combined in the same reaction tube.
This shows that the assay is capable to detect three genes in the same reaction tube. The amplification plots, the amplification efficiencies E , and R 2 score are represented in Fig 2.
The R 2 scores were determined as 0. The amplification plots a, c and the amplification efficiencies E b, d were represented. Then, the extracted RNA was used as template to test our assay. The comparative Ct performances of each assay were shown in Fig 3. The global increase in the COVID pandemic makes the development of faster, reliable, and sensitive virus detection methods a priority [ 21 ].
The assay is named as COV2-kit. Three different approaches were performed to optimize the protocol: simplex targets single gene , duplex simultaneously targets two genes and triplex simultaneously targets three genes.
The Ct values of the reactions are ranged in between 24 to In other words, the Ct value should not exceed 37 to accept the sample as positive. The Ct values in all tested approaches simplex, duplex, or triplex were under this threshold. The primers and probe for the RdRP gene were found to be more sensitive than these for the E gene. Thus, the samples with a low viral load can be detected by using these two sensitive gene targeting approaches. In addition to these viral genes, the reaction includes a human gene target, RP , as an internal control IC.
In general, the IC gene is tested in a separate tube for each reaction which decreases the sample size to be tested and increase the expenses. By using the current strategy, the number of reactions per sample is reduced. For instance, the assay allows testing 91 patients in well plates per run, thus provides less time and save expensive RT-PCR reagents.
COV2-kit revealed the Ct scores of the same gene as 27— CDC recommends the upper limit of the Ct as Accordingly, all tested samples were found below this limit by using the COV2-kit approach. Additionally, multiplex triplex rRT-PCR standardization test showed that this assay is efficient to detect three genes in the same reaction tube. Although RT-PCR tests are widely applied, and substitution tests are being developed, the current testing capability must meet new universal demands for fast, credible, and possible molecular detection [ 23 ].
In this method, we tried to bridge many challenges and weakness resulted in former assays and take benefits from their results and applications to improve novel assay. Compared to simplex or duplex analyses, the triplex analysis provides more accuracy and avoids possible negative false results that may be caused by a mutation in one of the viral genes [ 24 ].
It is an integral membrane protein functional in several biological processes of the virus such as assembly, budding, envelope formation, and pathogenesis [ 25 ]. Kakhki et al. They reported that ORF8 gene can be considered as a supplemental confirmatory test. Our study partly agrees with a protocol of a study published by Park et al.
They reported three-steps guidelines for designing and optimization specific primer sets: 1 the selection of primer sets for target genes RdRP , N , E , and S in SARS-COV-2 genome, 2 the in-silico effectiveness of primer and sequences of the amplicon, and 3 the optimization of PCR conditions.
This allows to run the reaction in a single tube. The described protocol which is like ours is wide-scale, high-integrity and accepted as gold standard. It will be applied to a larger number of viral extracted clinical RNA samples in subsequent tests to verify the specificity and sensitivity of the study.
Therefore, the first step of these protocols is centrifugation with subsequent lysis of the cell pellet. However, we need to recover free viral particles in solution, which do not sediment after routine centrifugation at 15, g.
For this reason we used the uncentrifuged sample directly mixed with lysis buffer, with subsequent precipitation of viral RNA in the whole mix volume.
Second, the acid pH method uses the anionic detergent Sodium dodecyl sulfate SDS that can lyse cells and viral coats through disruption of noncovalent bonds in proteins causing them to lose their native conformation Third, low pH and high concentration of salt make possible the selective recovery of RNA. Within the pH range of 5. RNA phosphodiester bond is more stable at acidic than alkaline pH, where it is susceptible to alkaline hydrolysis at pH greater than 6 Acid hydrolysis can only occur at pH lower than 2 12 , Therefore, it is essential to adjust the Lysis Buffer to pH 5, as described in materials and methods.
It is worth mentioning that all of the samples that changed their report had Cq values that were around the cutoff value of These changes occurred in both directions, meaning that some Cqs increased and some Cqs decreased. It would have been very clarifying to perform triplicated RNA extractions, in particular for undetermined samples, whose viral load is around the detection limit. Because of the above exposed information we consider the acid pH method robust and reliable.
In fact, it is currently being used in our diagnostic laboratory since the 3 rd week of April for routine detection of SARS-CoV2 in clinical samples.
This experimental procedure utilizes low cost reagents and equipment that can be found in standard molecular biology laboratories. The cost of extraction is a critical issue in most clinical laboratories, and the cost of our in-house method is around ten times lower than extraction kits.
Because of current environmental concerns, we would also like to highlight the lower plastic contamination generated by this in-house method.
Column-based extraction kits use several disposable tubes per sample, columns, bottles of buffer solutions, and plastic bags. Our in-house extraction method is by far, much more environmental friendly; it requires only two Eppendorf tubes per sample. Finally, our in-house method is comparable in hands-on time to commercial kits: it can be carried out in approximately 40 min for a set of 10 samples. However, it is important to mention that additional care must be taken in handling to avoid cross-contamination between samples.
This procedure can be a helpful alternative for laboratories facing supply-chain disruption and commercial kit shortages. Two types of biological samples were used. For preliminary evaluation of the RNA extraction methods we used saliva samples obtained from two asymptomatic volunteers. Saliva is routinely collected for the initial assessment of viral infection.
Two saliva samples were obtained from each volunteer and at least three independent RNA extractions were performed from each sample, obtaining a minimum of six RNA preparations to test each experimental procedure. Only one sample was obtained per patient: one portion of the sample was extracted using the High Pure Viral RNA Kit Roche , and another portion of the same sample was extracted using the acid pH method.
Samples were processed in the Laboratory of Diagnostic Microbiology of the same institution. All methods were performed in accordance with the relevant guidelines and regulations. The following experimental procedures were tested in this study. Saliva samples were centrifuged before taking an aliquot of supernatant for processing as described below.
The standard TRIzol-based method was evaluated 9 , 11 , The tube was mixed by inversion and incubated at room temperature for 10 min. Based on the procedure described by Plante et al. Then, 2. Under acidic pH, RNA can be separated from DNA and other molecules due to the differential polarity given by its hydroxyl groups, which maintains it in solution 12 , 22 , 25 , 26 , Samples were incubated on ice for 5 min and centrifuged at 15, g for 3 min at room temperature.
A new centrifugation step was made at 15, g for 5 min at room temperature. Supernatant was discarded and tubes were inverted in paper towel.
The pellet was dried, leaving the tubes open for 10 min. This RNA extraction method was considered as the gold standard for comparison purposes, and it is based in capture of RNA using columns with silica filters.
The RNase P gene is used as an internal control because many copies of it exist in the human genome, and it is readily detectable. The source of RNase P comes from the human cells that are present in every sample used. It is assumed that if human nucleic acids were extracted to detect the human gene RNase P, viral nucleic acids were also successfully extracted.
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