Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection

Gonzalez‐González, Everardo; Santiago, Grissel Trujillo‐de; Lara-Mayorga, Itzel Montserrat; Martínez-Chapa, Sergio O.; Álvarez, Mario Moisés

doi:10.1371/journal.pone.0237418

Cited by 31 publications

(25 citation statements)

References 34 publications

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“…Real-time qPCR thermocyclers are expensive instruments, which some testing centers have had to borrow from academic labs [ 26 ]. However, standard thermocyclers are relatively inexpensive and ubiquitous, and even less expensive, miniaturized PCR machines have been developed [ 45 ]. Visual inspection of images from a standard fluorescence gel imager was evaluated as a means of distinguishing the presence or absence of viral RNA without a real-time fluorescence readout.…”

Section: Resultsmentioning

confidence: 99%

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

et al. 2021

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Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.

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Section: Resultsmentioning

confidence: 99%

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

et al. 2021

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show abstract

“…Real-time qPCR thermocyclers are expensive instruments, which some testing centers have had to borrow from academic labs [26]. However, standard thermocyclers are relatively inexpensive and ubiquitous, and even less expensive, miniaturized PCR machines have been developed [45]. Thus, we tested whether it is possible to distinguish the presence or absence of viral RNA without a real-time fluorescence readout by visual inspection of images from a standard fluorescence gel imager.…”

Section: Endpoint Detection With a Fluorescence Imagermentioning

confidence: 99%

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Graham

Dugast-Darzacq

Dailey

et al. 2020

Preprint

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Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.

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“…But there are certain limitations in remote areas and portable diagnosis will be preferable using mini PCR based diagnosis kits. One study combined the mini PCR and multi-well plate reader for convenient and portable diagnosis under pandemic situation [16].…”

Section: Introductionmentioning

confidence: 99%

Development of RT-PCR Based Diagnosis of SARS-CoV-2

Patankar¹,

Zambare²

2022

Biotechnology to Combat COVID-19

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In the 2020, COVID-19 pandemic disease created an havoc situation world widely and mainly caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). It has been challenging task for researchers, scientists and medico-pharmaceutical organisations to find out rapid and reliable diagnosis methods. Among the all testing services, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the more accurate, rapid and authenticated molecular technique used for most of the diagnosis of major diseases. It has been a global priority to fix the rapid diagnosis method to combat against the pandemic COVID-19. Thus, the present chapter mainly focussing on the progress of RT-PCR method development though various processes of data collection on isolation of whole genome sequence, its primer and method designing. In this scenario, India suddenly become the global leader for vaccine development and hence the challenges and RT-PCR kit development in India and rest of the world has been be discussed. World wide many Government and private agencies and industries have taken an initiative for diagnosis of SARS-CoV-2 hence this chapter also summarised the scope of RT-PCR to combat pandemic situation in future.

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Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection

Cited by 31 publications

References 34 publications

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Development of RT-PCR Based Diagnosis of SARS-CoV-2

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