COVID-19 PCR Molecular Diagnosis


A new strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has recently been identified as an infectious disease affecting the respiratory system of humans. This disease is caused by SARS-CoV-2 which was identified in Chinese patients with severe pneumonia and flu-like symptoms. COVID-19 is a contagious disease that spreads rapidly through droplet particles that arise through the sneezing and coughing action of an infected person. Reports of asymptomatic carriers changed the scenario of symptom-based diagnosis in COVID-19 and intensified the need for proper diagnosis of the majority of the population to combat the rapid transmission of the virus.

The diagnosis of positive cases is necessary to guarantee timely care for affected people and also to stop the further spread of the infection in the population. Collecting samples at the right time and from the exact anatomical site is crucial for proper molecular diagnosis. After the complete genome sequence became available, China formulated RT-PCR as a primary diagnostic procedure to detect SARS-CoV-2. Many in-house and commercial diagnostic kits have been or are in development that has the potential to reduce the diagnostic burden on primary diagnostic techniques such as RT-PCR. Serologic diagnosis is another broad category of tests that can detect different serum antibodies such as IgG, IgM, and IgA in an infected patient.

The PCR-based diagnostic procedures that are commonly used for pathogen detection need sophisticated machines and the assistance of a technical expert. Despite their reliable accuracy, they are not cost-effective tests, which a common man can afford, so it becomes imperative to search for other diagnostic approaches, which could be cost-effective, fast, and sensitive with consistent accuracy. To make such diagnostics available to the common man, many techniques can be exploited, which are Point of Care (POC), also known as bedside testing, which is being developed as a promising and portable tool in diagnostics. of pathogens.

Other Lateral Flow Assay (LFA)-based techniques such as SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 Publisher Limited Uniform Detection Assay (FELUDA), etc. have shown promising results in the rapid detection of pathogens. Diagnosis is of critical importance in the pandemic situation when there is no potential drug for the pathogen available on the market. This review summarizes the different diagnostic approaches designed or proposed to combat the widespread diagnostic crisis due to the sudden outbreak of a new pathogen, SARS-CoV-2 in 2019.

Sampling and biosafety measures

High levels of upper and lower respiratory tract viral loads have been shown in COVID-19 patients within 5 to 6 days of symptom onset (Pan et al., 2020). For an early diagnosis of COVID-19, nasopharyngeal or oropharyngeal swabs are recommended (Chan et al., 2004) (Zou et al., 2020), however, a single nasopharyngeal swab is a method of choice for healthcare professionals because patients can easily tolerate it and it is safe to handle. To obtain an adequate nasopharyngeal swab sample, the swab must penetrate deep into the nasal cavity causing tears in the patient (Druce et al., 2012).

Collected swabs should be transported immediately using transport media to the diagnostic laboratory, ideally under refrigerated conditions (Druce et al., 2012). Patients with severe COVID-19 pneumonia have shown high viral loads in bronchoalveolar lavage fluids; however, nasopharyngeal swabs were not compared in the particular study (Wang et al., 2020). These patients have also shown high levels of viral RNA in faecal samples (Zhang W. et al., 2020). Therefore, the preferred method for collecting samples from advanced COVID-19 patients is from stool or rectal swabs (Cheng et al., 2004).

For the safety of healthcare professionals and the proper processing of samples, it becomes imperative to take maximum precautions when collecting, transporting, and processing COVID-19 samples. In response to this, healthcare professionals should wear goggles, N95 respirators, gloves, full-sleeved gowns, or PPE kits to minimize direct contact with COVID-19 positive patients (Karthik et al., 2020).

During the early days of the COVID-19 pandemic, a simple and convenient approach to collecting patient samples was a bold necessity to replace the painful process of collecting nasopharyngeal swabs. In that scenario, the Rutgers Clinical Genomics Laboratory came up with the idea of ​​developing an RT-PCR-based technique, which can detect SARS-CoV-2 RNA in self-collected saliva samples. They developed an assay kit, which was marketed as the TaqPath™ COVID-19 Combination Kit. This strategy reduced the risk of infection during sample collection by health professionals (Afzal, 2020). Sample collection for protein-based diagnoses such as IgG/IgM and LFA requires blood samples from patients.

RT-PCR as a first-line diagnostic method for the diagnosis of COVID-19

Molecular diagnostic approaches are appropriate compared to other syndromic testing approaches because molecular diagnostics target the genome or proteome of the pathogen, making it a specific and reliable diagnostic method (Zhou et al., 2020b). For a new pathogen, sequencing and diagnosis become essential to recognize the nature of the pathogen and its genomic composition. Random amplification and deep sequencing strategies played a critical role in the early identification of SARS-CoV-2, which was confirmed as a member of the coronavirus family through different bioinformatics approaches (Briese et al., 2014).

Using metagenomic sequencing, the first genomic sequencing for SARS-CoV-2 was performed (Miller et al., 2019; Sheridan 2020a). On January 10, 2020, the findings were made public and the sequences were submitted to the GenBank Sequence Repository (Wu et al., 2020). The publication of the complete genome sequence of SARS-CoV-2 in public databases made it easier for scientists to design primers and probes for laboratory diagnostics of COVID-19 (Corman et al., 2020). After the identification of this virus, the WHO recommended real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), which is a nucleic acid-based technique, as the first-line diagnostic approach. to detect SARS-CoV-2 infection in suspected patients.

RT-PCR is highly sensitive and can detect infection at minute levels of pathogens present in the patient sample. It is a nucleic acid-based technique used to amplify a target gene/nucleotide present in a sample, helping to detect a specific pathogen and discriminate it from other related pathogens. There are generally two possible ways to perform RT-PCR, including the one-step assay or the two-step assay. The one-step assay consolidates reverse transcription and PCR amplification in a single tube, making the detection process fast and reproducible; however, this assay provides a lower target amplicon generation.

In the case of a two-step assay, the reactions are performed sequentially in two separate tubes, which makes it a time-consuming, but sensitive assay compared to the one-step assay format (Wong et al. Medrano, 2005). Although eleven nucleic acid-based protocols and eight antibody detection kits have been approved by the National Medical Products Administration (NMPA) in China, PCR was considered as a preferred diagnostic technique. The US Centers for Disease Control and Prevention (CDC) uses a one-step PCR format to diagnose COVID-19. The assay is performed by isolating RNA from the sample and adding it to the master mix containing forward and reverse primers, nuclease-free water, reaction mix (reverse transcriptase, polymerase, nucleotides, magnesium, and other additives).

A PCR thermocycler is loaded with the extracted RNA and master mix, and the temperature is set to run the PCR reaction. Cleavage of a fluorophore quenching probe during this reaction generates a fluorescence signal that is detected by the thermocycler and the progress of amplification is recorded. Positive and negative controls should be included whenever an RT-PCR reaction is performed, making interpretation of results easy and strict (Chan et al., 2020). RT-PCR and some biosensor-based diagnostic kits can detect SARS-CoV-2 nucleotides in faecal samples or wastewater that may be a warning of an infectious disease outbreak in a particular area.

SARS-CoV-2 can survive from hours to days in untreated wastewater (Orive et al., 2020). RT-PCR is a sensitive and rapid screening tool in molecular diagnostics. It can detect and amplify even a few copies of a specific genomic sequence in a variety of samples but depends on certain aspects to provide reliable results, such as proper sample collection, transport, storage, and processing (Afzal, 2020). It has been used for the detection of various viruses such as adenoviruses, rotaviruses, astroviruses, and many enteric viruses isolated from faecal samples (Kowada et al., 2018).

A major drawback of this technique is the need for a well-equipped laboratory and technical staff to manage the experiment, which cannot mitigate the increasing demand for rapid tests during pandemic situations such as COVID-19 (Bustin and Nolan, 2004). RT-PCR-based kits are very expensive and take a long time to deliver results, making it essential to seek other rapid and reliable diagnostic methods (Hofman et al., 2020; Sheridan 2020b).