This presentation highlights the methods used in recombinant DNA technology to detect the presence of the novel coronavirus in patient samples.

2.  Large family of single-stranded RNA viruses
 Betacoronavirus
 Positive sense ssRNA
 Spike (S) glycoproteins promote entry into
cells
 Interact directly with angiotensin-converting
enzyme 2 (ACE2)
 Unexpected furin cleavage site at the
S1/S2 boundary of SARS-CoV-2 S, which is
cleaved during biosynthesis
SARS-CoV-2
STRUCTURE
SARS-CoV-2 has four structural proteins:
the E and M proteins - which form the viral
envelope; the N protein - which binds to the
virus’s RNA genome and the S protein -
which binds to human receptors.
The nonstructural proteins get expressed
as two long polypeptides. This group of
proteins includes the main protease (Nsp5)
and RNA polymerase (Nsp12).

3. DETECTION OF SARS-
CoV-2
 Reverse transcription polymerase chain reaction
 Digital droplet PCR
 Reverse Transcription - Loop-mediated isothermal amplification
(RT-LAMP)
 Transcription Mediated Amplification (TMA)
 CRISPR based assay
 CovidArray

4. POLYMERASE CHAIN REACTION
AND MECHANISM
 simple, widely used technique, highly sensitive, gives result in a
few hours.
 for the amplification and detection of DNA and RNA sequences.

5. REVERSE TRANSCIPTASE PCR
AND REVERSE
TRANSCRIPTASE ENZYME
QUANTITATIVE PCR
(qPCR)/ (rtPCR)
 Quantitative PCR detects,
quantifies the amount of nucleic
acid. Fluorescent labeling enables
the collection of data as PCR
progresses, i.e. in real time.
RT-qPCR
 For SARS-CoV-2, the one-step
method is preferred due to its quick
set up, limited sample handling,
reduced bench time and less
chances of cross contamination.

6. MECHANISM FOR
DETECTION OF SARS-
CoV-2
CYCLE THRESHOLD
VAULE (Ct)
 Ct refers to the number of cycles
needed to amplify viral RNA to reach
a detectable level, i.e. how may
cycles does it take for the viral RNA
to be detected.
 A person having a very high viral
load will show a much less Ct value
as compared to someone with little
viral load.

7. DIGITAL DROPLET PCR
 Although RT-PCR technique is considered the gold standard for
SARS-CoV-2 detection, it does come with a few shortcomings –
such as sample collection, viral lysis and RNA purification,
shortages in laboratory supplies and RNA extraction kits.
 Digital droplet PCR facilitates quantification of target nucleic
acid sequences.
 It enables accurate SARS-CoV-2 RNA quantification from crude
lysate without nucleic acid purification. It provides absolute
viral counts from crude lysate, preventing the need for a
standard curve.

8. DD-PCR MECHANISM FOR SARS-COV-2
DETECTION
(A) A mixture containing dNTPs, primers, and probes is prepared for the amplification reaction.
(B) Water-in-oil droplets are generated using a microfluidic flow-focusing system.
(C) The generated droplets and oil are collected in PCR tubes.
(D) PCR amplification occurs in each droplet.
(E) Droplets are checked by a photoelectric detection system composed of lasers and
photomultiplier tubes.
(F) Finally, the fraction of positive droplets is fitted to the Poisson distribution to determine the
absolute copy number of target molecules in the. original reaction mixture.

9. APPLICATIONS IN THE
PANDEMIC
High-sensitivity detection of SARS-CoV-2
Evaluation of the viral load in different clinical sample types
Evaluation of sample preparation methods
Dynamic monitoring of disease progression
Preparation of nucleic acid reference materials
Monitoring the virus concentration in the environment
Detection of SARS-CoV-2 mutations
Evaluation of anti-SARS-CoV-2 drugs
ADVANTAGES
 Absolute amplification
 Higher sensitivity
 Better stability
 Higher precision

10. REVERSE TRANSCRIPTION - LOOP
MEDIATED ISOTHERMAL
AMPLIFICATION
 alternative to RT-qPCR.
 nucleic acid amplification
occurs at one temperature.
 allows quicker assay, easy
to use, more cost effective
than qRT-PCR assays.
 maintains specificity and
sensitivity parameters.

11. LAMP MECHANISM
 repetitive process
 based on formation of loop structures
 amplification of the loops via specific primers

12. MECHANISM FOR SARS-CoV-2
DETECTION
viral RNA converted to cDNA.
 A set temperature (either 63°C or 65°C) is maintained.
4 sets of LAMP primers targeting different gene regions of SARS-CoV-2 are used.
A colorimetric detection differentiates the positive and negative samples. Positive samples are
determined by a color change from cresol red to an orange-yellow color within 30 min after
the start of the incubation at 65°C.

13. TRANSCRIPTION MEDIATED
AMPLIFICATION
 single tube, isothermal amplification technology used to amplify specific regions of
either RNA or DNA more efficiently than RT-PCR.
 uses a retroviral reverse transcriptase and T7 RNA polymerase.
 Viral RNA target is hybridized to a specific
capture probe and additional oligonucleotide
containing a T7 promoter primer.
 reverse transcription into cDNA occurs.
 An additional primer generates a dsDNA,
which is transcribed into RNA amplicons by
T7 RNA polymerase.
 Detection via single-stranded nucleic acid
torches.
MECHANISM OF TMA FOR SARS-CoV-
2 DETECTION

14. CLUSTERED REGULARLY INTERSPACED
SHORT PALINDROMIC REPEATS
(CRISPR)
 takes advantage of the collateral cleavage
activity of Cas endonucleases
 followed by isothermal amplification of the
target
 results can be read using paper strips to
detect the presence of the SARS-CoV-2
virus without loss of sensitivity or specificity.
Two methods –
A . SHERLOCK method uses Cas13 that
can excise reporter RNA sequences in
response to activation by SARS-CoV-2-
specific guide RNA.
B . The DETECTR assay that relies on the
cleavage of reporter RNA by Cas12a to
specifically detect viral RNA sequences of
the E and N genes of the virus.

15. THE SHERLOCK METHOD
 Viral RNA is extracted from the
clinical sample and amplified
using reverse transcription.
 The T7 promoter sequence is
added.
 The Cas13a binds and
becomes active.
 cleaves both the target RNA
and the reporter.
 The signal can be detected
using fluorescence or lateral
flow-based readout methods.

16. COVIDARRAY MECHANISM
 first DNA microarray-based assay to detect viral genes in the swabs
 assay to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs.
 based on solid-phase hybridization of fluorescently-labeled amplicons.
 has high sensitivity.
 allows the reduction of amount of false-negative results and the total analysis time
to about 2 hours.

17.  https://cen.acs.org/biological-chemistry/infectious-
disease/know-novel-coronaviruss-29-
proteins/98/web/2020/04
 https://www.sciencedirect.com/science/article/pii/S
1201971220301235
 Walls, A. C., Park, Y.-J., Tortorici, M. A., Wall, A.,
McGuire, A. T., & Veesler, D. (2020). Structure,
Function, and Antigenicity of the SARS-CoV-2
Spike Glycoprotein.
Cell. doi:10.1016/j.cell.2020.02.058
 https://pubs.acs.org/doi/10.1021/acscentsci.0c005
01
 https://www.fda.gov/media/139279/download
 https://www.aacc.org/cln/cln-
stat/2020/december/3/sars-cov-2-cycle-threshold-
a-metric-that-matters-or-not
 https://www.medpagetoday.com/infectiousdisease/
covid19/90508
 https://www.nature.com/articles/s41598-020-
80715-1
 https://onlinelibrary.wiley.com/doi/10.1002/VIW.202
00082
REFERENCES