Development and validation of an in-house, low-cost SARS-CoV-2 detection assay

Abstract

Background One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green–based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol. Methods We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green–based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R 2 = 0.89). Conclusions These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.

Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid

“…Moreover, despite the required adaptation of this methodology, it is still worthy due to the low cost of this assay. The SYBR Green qPCR multiplex is supposed to cost US$ 15–20 as observed in the literature [ 21 , 22 ]. Here, it was estimated < 1 dollar considering only master mix and primers; whereas qPCR multiplex using three different probes, it was estimated the cost of US$ 8–15, considering commercial kits with master mix, primers, and in this case probes, available for molecular diagnostic of other infectious diseases.…”

Descriptive comparison of ELISA and PCR methods in SARS-CoV-2 detection

“…Sensitivity of the assay was consistent with the 5 other assays, which was 98.2%, indicated by the successful detection of 54 positive cases out of 55 across all assays in the comparison. Two months after, a SYBR green-based RT-PCR was developed by Alhamlan et al with primer targets consisted of assay consisted of RdRp sequence, as well as structural protein genes of SARScov-2 [13]. The validation of the assay was carried out on 190 nasopharyngeal swabs collected from hospitalized patients.…”

A low-cost molecular test for SARS-CoV-2 detection suitable for variant discrimination and community testing using saliva

“…One advantage of these tests, as opposed to RT-PCR with fluorogenic probes, is that they allow for assessment of the amplification specificity by simply examining the amplicon melting curves at the end of the PCR [18]. While most SYBR Green-based RT-PCR protocols have been established for use with RNA isolated from naso-or oropharyngeal specimens [11,16,[19][20][21][22][23][24][25][26][27], there is promising evidence for the use of saliva samples with this type of tests. Ganguly et al amplified synthetic SARS-CoV-2 RNA spiked into saliva specimens from healthy donors [28].…”