1.1. High-Throughput PCR-Based Detection

In the current report, we are highlighting a PCR-based COVID 19 detection strategy. Such tests use an approach to identify presence or absence of genetic material(s) of the virus in the samples. This PCR technique amplifies small stretch of viral genome using custom-designed “primers” at a high stringency condition. (a) The sample is collected from the oral/nasal cavity of suspect patients using a synthetic fiber swab with plastic shafts; (b) transit the swabs from the collection point to the testing point should be done in tubes containing viral transport media; (c) high-throughput RNA extraction from the swab samples; (d) cDNA synthesis of the extracted RNA in 96-well plate format; (e) 96-well plate PCR using selective primers using 10 picomoles of each forward and reverse primers; (f) capillary electrophoresis of the 96-well PCR plate to identify PCR products in positive samples. Primer pairs for three of more than three genes may be used in parallel amplification for statistical validation. Figure 1 depicts a schematic representation of the overall procedure for COVID 19 detection using PCR technology.

1.2. Reagents and Kits

As explained in Figure 1, the first step is the collection of samples on swab from the throat or nasal cavity in viral transport media (TMMEDIA, Delhi, India). The kit has one sterile flocked collection swab and a tube of Viral Transport from the place of collection to testing laboratory. Buffers and antibiotics content of the medium are recommended by Centers for Disease Control and Prevention (CDC) and WHO to ensure the violability of the coronavirus during transport. The next step is the extraction of RNA from viral transport media in a 96-well plate format. High-throughput RNA extraction kits, such as PureLink™ Pro 96 total RNA Purification Kit (Thermo Fischer), E-Z 96^® Total RNA Kit (Omega Bio-Tek), Mag-Bind^® Total RNA 96 Kit (Omega Bio-Tek), and 96-well purification of total RNA from cells (Qiagen) could be used for this purpose. The third step is RT-PCR of the extracted RNA in a 96-well format using SuperScript™ III First-Strand Synthesis System (ThermoFischer) or TurboCapture 96 mRNA kit (Qiagen). The forth step is 96-well PCR using the designed RIL-CoVID primers (Table 1) and 2× QIAGEN Multiplex PCR Master Mix (Qiagen) or AmplyMAX™ Multiplex PCR Master (Canvaxbiotech). PCR machines with 96-well thermal block of ThermoFischer, Biorad, or any other model could be used for PCR amplification. The detection of desired PCR products in 96-well plate could be done using 7100 CE System (Agilent) or Capillary Electrophoresis (SCIEX) as detailed in Figure 1.

1.3. Designing of Primers for SARS-CoV-2

Genome of 41 variants of SARS-CoV-2 was downloaded from NCBI and aligned using multiple sequence alignment (MSA) tool for phylogenetic analysis using MEGA 7.0 software package [4]. The phylogenetic tree constructed using 28.5 KB consensus DNA region indicated a high degree of similarities among the genomes of the various SARS-CoV-2 sequences at different part of the world (Fig. 2). We have identified some conserved regions from MSA alignment file and have designed PCR primers using an in-house algorithm. A set of 100 pairs of oligos were then subjected to similarity searches against other viral genomes and human genome. Finally, the primers which showed less than 50% homology in human genome and 100% similarity with the genome of SARS-CoV-2 were analyzed further. Finally, the selected primers and probes were also subjected to similarity searchers with the genome of other respiratory viruses available in non-redundant database. The unique primers which selectively binds to the genome of SARS-Cov-2 were selected. Table 1 has the list of primers and probes which can be used for PCR amplification and hybridization. The DNA fragments amplified using the PCR primers were identified as genes (partial) for surface glycoproteins, envelope protein “E”, membrane glycoprotein “M”, ORF3a protein and nucleocapsid phosphoprotein “S” (Table 1). Except ORF3a, all these proteins fall in the category of structural proteins which plays an important role in the assembly of virion capsid.

Figure 1: Schematic diagram showing a process flow for PCR-based early detection of SARS-Cov-2 in throat/ nasal swab samples. [Click here to view]

Table 1: List of RIL-COVID primers and probes for designing PCR or FISH based approach for detection of virus.

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1.4. Community Infection Assay

The PCR method discussed here could also be used for scoring stage-3 of epidemic or community screening of infection by SARS-CoV-2. Pooled cDNA (mix of 10–12 samples) could be added in each well of PCR plate. After PCR amplification capillary electrophoresis of each well could be done to identify the well containing infected samples. During stage 1 and stage 2 of an epidemic, majority of samples tested (95%) are negative which results loss of reagents and resources. Therefore, a method that does not overload the qRT PCR with array of samples is desirable. This will also negatively impact the throughput of screening. The process described here (Fig. 3) will help in identifying uninfected sample pool and thereby the requirement of qRT-PCR-based high-value and efficient systems could be employed to the focused samples as a confirmatory test. The method will also help in driving a super-high-throughput assay to score community transmission with a statistically validated model [5] for heavy density population, such as slum-dweller in Mumbai or migrant workers.

We hypothesize that the abundance of the mRNAs of the genes listed in Table 1 will be high at the early days of infection, and therefore, nucleic acid-based detection system will be more efficient. On the other hand, IgG- or IgM-based immunological principles may miss the window of the early detection as the titer of these proteins will reach the detectable limit in the blood after 4–10 days of infection (asymptomatic stage). Therefore, we have focused on developing process and designed probes and primers for the early detection of infection (1–4 days). In the Table 1, we have also listed probes which could be used for in situ hybridization assay [6]. The stringency of the forward and reverse oligonucleotides/PCR primers were checked in silico for potential secondary structure formation and primer dimerization using various in silico PCR tools available online. In the pipeline, we have added capillary electrophoresis to reduce time lag and increasing throughput of analysis. However, in the absence of such high-throughput capillary system agarose gel electrophoresis could also be used and gel loading with multichannel pipettes will be of help. Although, such manual intervention will reduce throughput but pooled sample analysis described in Figure 3 will still facilitate large-scale sample analysis. Kits and methods which work best for the proposed pipeline are derived by talking to more than 100 scientists working across different labs of India. However, kits with similar specifications could also be used based on the availability at different countries.

Figure 2: Neighbor-joining phylogenetic tree derived using 41 genome of SARS-CoV2 showing very high similarity between genomes of the virus sequenced in different countries. The tree was constructed with MEGA 7.0 software package and used the bootstrap value for 500 replications. Genome of MT226610 SARS-CoV-2/ KMS1/CHN was used to root the tree. The bar represents 1 base changes in 10,000 nucleotides. [Click here to view]

Figure 3: Schematic representation of super-high-throughput screening for COVID 19 infection using pooled-sample analysis. The method proposed here enables 1,000 samples analysis in one shot for stage-3 or community infection scoring. [Click here to view]