In a new study published on the preprint server medRxiv*, researchers from the Netherlands develop an ultrafast detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by direct reverse-transcriptase polymerase chain reaction (RT-PCR) without RNA extraction that is suitable for large-scale use. Herein, the. researchers describe the high-efficient method and validate it against a reference quantitative-PCR (qPCR) method to rapidly test SARS-CoV-2 in clinical samples.
Study: Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach. Image Credit: Salov Evgeniy / Shutterstock.com
The need for rapid and easy-to-implement diagnostic methods, specifically nucleic acid detection methods, cannot be understated during the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2. The current gold standard for testing for the presence of SARS-CoV-2 is the nucleic acid amplification tests (NAAT), such as multiplex RT-PCR. This test is highly sensitive, specific, and rapid in detecting the virus.
However, it is a challenge to have a method that is quick, sensitive, and reliable, along with an option for large-scale SARS-CoV-2 screening applications. In the present study, the researchers have shown a highly efficient direct RT-PCR protocol using their proprietary NextGenPCR ultra-fast thermocycler for SARS-CoV-2 genomic targets detection in nasopharyngeal swabs (NPS) without the need to perform RNA extraction.
Notably, according to the United States Centers for Disease Control and Prevention (CDC) Interim Guidelines for Collecting and Handling of Clinical Specimens for COVID-19 Testing, NPS are the preferred biological samples for SARS-CoV-2 detection.
NextGenPCR direct RT-PCR method
The proposed method in the current study is based on multiplex RT-PCR amplification of three target sequences followed by end-point detection of amplicon-specific probe fluorescence. The three sequences are SARS-CoV-2 N1 and ORF1ab gene sequences from the virus, as well as a sequence in the human RPP30 gene.
In the reaction, FAM-labelled (fluorescein amidites) oligonucleotide probes are used to detect SARS-CoV-2 sequences, whereas Cy5- labeled oligonucleotide probes are used to detect the human sequence.
The use of a dual probe design is highly advantageous. Both amplicons contribute to the increased signal amplitude in end-point detection. Also, they provide strong analytical protection against variation of target sequences, as observed with emerging SARS-CoV-2 variants.
The researchers probed the human sequence to also confirm whether the PCR reaction is occurring without any inhibition and to indicate the presence of human genetic material in sample wells. This helps to verify correct sampling and pipetting by the personnel.
Using extensive bioinformatical analyses and in silico PCR, the researchers designed the primer and probe oligonucleotide sequences to minimize amplification of off-target sequences and formation of primer-dimers or other secondary structures.
The researchers collected NPS samples from 101 individuals who were suspected of SARS-CoV-2 infection at the Canisius Wilhelmina Hospital (Nijmegen, The Netherlands). The NPS were directly transferred to a sterile microtube containing AMIES liquid and heat-inactivated at 100°C for 10 minutes, after which an aliquot was taken for RNA extraction and qPCR measurements, and the remaining volume was used for the novel analytical method.
The researchers heat-sealed the reaction mix containing the RT-PCR Chemistry 2x, Primers, and Probes with the samples on a NextGenPCR Semiautomatic Heat Sealer and transferred the product to a NextGenPCR machine for thermal cycling. After PCR was completed, the sealed microplate was placed on an imaging anvil, transferred to a FLUOstar Omega Microplate Reader, and scanned to obtain the fluorescence readout results for data analysis.
Further, for the measurements of synthetic RNA, the researchers used the Bio-1000F Gel Imager to scan the microplate and interpret the results using custom-designed QuickDetect software. The SARS-CoV-2 qPCR detection method was used as a reference method.
The researchers reported that the direct RT-PCR method on synthetic RNA dissolved in water or AMIES liquid showed similar amplification linearity and limit of detection. They also reported that for the SARS-CoV-2 detection in a panel of surrogate clinical samples, direct RT-PCR provided accurate and sensitive results, even without prior nucleic acid extraction. The researchers reported that the analytical sensitivity of the developed assay was determined at 1.0 × 100 copies/ul.
The present study established that direct RT-PCR has high clinical sensitivity, specificity, and sample calling agreement as compared to reference qPCR methodology.
The researchers elaborated that this method took about 27 minutes/sample from sampling to end-result, while the reference qPCR method takes about 60 minutes/sample, the latter of which is a high turnaround time. Because of its superior speed, the researchers argue that this novel method is suitable for large-scale screening purposes where time to detection is a crucial factor.
The researchers confirmed the absence of any cross-reactivity with other respiratory viruses. They tested the method in 14 clinical samples positive for respiratory viruses other than SARS-CoV-2, thus confirming NextGenPCR primer-probe specificity.
The present study demonstrated a screening method for the detection of SARS-CoV-2 viral particles in human clinical samples through direct RT-PCR with significantly reduced time and without the need for sample lysis or prior extraction of RNA. As compared to the reference qPCR method on extracted RNA, this is a highly accurate method to identify SARS-CoV-2 positive and negative samples by end-point NextGenPCR RT-PCR.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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