AU2021103139A4 - Differential detection kit for common SARS-CoV-2 variants in COVID-19 patients - Google Patents

Differential detection kit for common SARS-CoV-2 variants in COVID-19 patients Download PDF

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AU2021103139A4
AU2021103139A4 AU2021103139A AU2021103139A AU2021103139A4 AU 2021103139 A4 AU2021103139 A4 AU 2021103139A4 AU 2021103139 A AU2021103139 A AU 2021103139A AU 2021103139 A AU2021103139 A AU 2021103139A AU 2021103139 A4 AU2021103139 A4 AU 2021103139A4
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Sorayya Ghasemi
Leila Mousavizadeh
Marcel Mozafari
Milint Neleptchenko Wintrasiri
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Mozafari Marcel Reza Prof
Mousavizadeh Leila Dr
Ghasemi Sorayya Dr
Neleptchenko Wintrasiri Milint Mrs
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Mousavizadeh Leila Dr
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Abstract

The invention relates to a differential detection kit for common SARS-CoV-2 variants in COVID-19 patients, the kit comprising a set of probes and primers specific for spike gene mutations, as well as to the nucleocapsid gene and the RNA-dependent RNA polymerase gene. Detection using real time PCR methods are disclosed using probes with different colors for different mutations.

Description

Title of the invention:
Differential detection kit for common SARS-CoV-2 variants
in COVID-19 patients
Inventors:
Marcel Reza Mozafari 1, Sorayya Ghasemi 2, Milint Neleptchenko Wintrasiri 3
, and Leila Mousavizadeh4
Affiliations of Inventors:
1. Australasian Nanoscience and Nanotechnology Initiative (ANNI), Monash University
LPO, Clayton, Victoria 3168, Australia. dr.m.r.mozafari@gmail.com
2. Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord
University of Medical Sciences, Shahrekord, Iran. sorayya.ghasemi@gmail.com
3. Supreme NanoBiotics Co. Ltd. and Supreme Pharmatech Co. Ltd., 399/90-95 Moo 13
Kingkaew Rd. Soi 25/1, T. Rachateva, A. Bangplee, Samutprakan 10540, Thailand.
milint@supremepharmatech.org
4. Department of Virus-host Interaction, Heinrich-Pette-Institut (HPI), Martinistrasse 52,
20251 Hamburg, Germany. lmosavi2007@yahoo.com
DESCRIPTION
[0001] The embodiments herein relate to a diagnostic kit for identifying virus
variants in COVID-19 patients.
[0002] The present invention more particularly relates to new types of probe
and primer designs for accurate identification of SARS-CoV-2 variants.
[0003] The mixture of primer-probe in this kit is designed based on the six
target gene, which targets the specific conserved sequence encoding the RdRp
(RNA-dependent RNA polymerase) region and the nucleocapsid protein N region
as well as detection of different variants based on specific mutations (N501Y,
L452R, D614G, P681H), which have been separated into two segments, such as
two vials.
BACKGROUND OF THE INVENTION
Technicalfield
[0004] Coronavirus is a parasitic organism containing single-stranded RNA,
of positive polarity, with the size of approximately 30 kilobases that replicates in
the cytoplasm of the host cells; being Human cells or animal cells. The 5' end of
the genome of Coronavirus has a capped structure and the 3' end contains a polyA
tail. This virus is enveloped and comprises, at its surface, peplomeric (spike)
structures known as spicules.
[0005] The N protein or nucleocapsid protein (45-50 kDa) that is the most
conserved among the coronavirus structural proteins is necessary for encapsulating
the genomic RNA and also for directing its incorporation into the virion. It is
known that this protein is also involved in the replication of the RNA.
[0006] When the host cell is infected, the open reading frame (ORF) situated
at the 5' of the viral genome is translated into a polyprotein that is cleaved by the
viral proteases, and consequently releases several nonstructural proteins such as the
RNA-dependent RNA polymerase and the ATPase helicase (Hel). These two
proteins are involved in the replication of the viral genome and in the generation of
transcripts that are employed in the synthesis of the viral proteins. The mechanisms by which these subgenomic mRNAs are produced are not completely elucidated.
Nevertheless, recent evidences indicate that the sequences for regulation of
transcription at the 5' end of each gene represent signals which regulate the
discontinuous transcription of the subgenomic mRNAs (1).
[0007] The proteins of the viral membrane (S, E and M proteins) are inserted
into the intermediate compartment, whereas the replicated RNA (+ strand) is
assembled with the N (nucleocapsid) protein. In a later stage, this protein-RNA
complex combines with the M protein contained in the membranes of the
endoplasmic reticulum of the host cell. The viral particles form when the
nucleocapsid complex buds into the endoplasmic reticulum. The virus then
migrates across the Golgi complex and eventually leaves the cell (e.g., by
exocytosis). The site of attachment of the virus to the host cell is at the level of the
spike protein (S protein).
[0008] Amplification of RNA-dependent RNA polymerase (RdRp), N
protein, internal control, and two mutant variants (N501Y, L452R) can be
qualitatively monitored by the increasing fluorescence signal detected by a Real
time PCR instrument. However, in the second vial reaction buffer is able to detect
two other mutant variants (D614G, P681H). The PCR detection system includes an
internal control primer-probe. The cycle threshold (CT) results / values of the
internal control provide the accuracy of sampling and extraction process. This is in order to avoid false-negative results.
DESCRIPTION OF THE RELATED ART
[0009] Since 2002, we have faced several outbreaks of coronavirus strains in
the world. SARS-CoV-2 affected around 800 people in 2002 which led to the death
of 10 percent of the infected individuals. On the other hand, MERS-CoV affected
around 2300 people in 2012 with a 35 percent death rate.
[0010] The third outbreak of this family of viruses is the pandemic of SARS
CoV-2 (i.e., the cause of COVID-19 disease) in 2019 and 2020, with 9.678.494
confirmed cases and a 2.72 percent mortality rate. Lack of safety-related
experience from confrontation with this virus has led to the susceptibility and
sensitivity of people to this infection (2).
[0011] SARS-CoV-2 is a positive-strand RNA virus with four main structural
proteins including spike, envelope, membrane, and nucleoprotein. Spike protein (S
protein) plays a key role in the attachment of the virus to the receptor of the host
cell (ACE2). S protein consists of S subunit (i.e., receptor-binding subunit) and
S2 subunit (i.e., membrane fusion subunit). S1 subunit contains a receptor-binding
domain (RBD) which is responsible for the attachment of the virus to the cellular
receptor (3).
[0012] The mutation of coronavirus varies from moderate to intense. The
most common recombinant mutation of coronavirus occurs in the S protein
expressing gene located at the RBP section and leads to the change of amino acid
sequence of the part which is required for the attachment to the host cell.
[0013] Recently, in the early stages of the COVID-19 pandemic, 149
mutations were detected in 103 gene sequences which have led to the identification
of the two mainstream mutations called L and S. The number of mutations is
increasing with the progression of the pandemic. More detailed reviews on the
characteristics of these strains can help assess the potential risks, development of
the treatment methods, and preventive strategies.
[0014] Still, it is not clear that these mutations contribute to the deterioration
of the disease or intensity of the virus transmission, as it has been shown that these
mutations can occur in genomic parts that do not affect the characteristics of the
virus. Although, some evidence indicates that the D614G strain can enhance the
infectivity of COVID-19.
[0015] Recently, B.1.1.7 (a new mutated strain of SARS-CoV-2 emerged in
England) indicated a 70 percent higher transmission rate in comparison to the wild
type. This mutation has occurred at the N501Y locus which translates to the
surface of the spike protein. Although this mutation has not affected the
vaccination, it appears that it has enhanced the transmission rate and it is less likely to be detected by the Real time-PCR technique.
[0016] N501Y mutation is one of the main six sequences of the RBD and it
was revealed by prior studies that this mutation can enhance the ability of the
attachment to ACE2 in human and mouse models. N501Y is associated with the
increase of infectivity and virulence in the mouse model.
[0017] In addition to this mutation in coronavirus, there is another mutation
called P681H which is one of the main four sequences of the viral entry and causes
a furin cleavage site between S Iand S2 in the S protein. This region was not
observed in other coronaviruses, and it facilitates virus entry to the epithelial cells
of the respiratory system and virus transmission in the animal model.
[0018] Two other strains which are created by mutations of the spike protein
are K417N and E484K, which cause viral transformation and can affect the
vaccines based on the primary type of the virus. However, recent studies revealed
no indication of a decrease in the antibody levels after the administration of the
Pfizer vaccine.
[0019] One study on the current mutations including N501Y, A570D,
D614G, P681H, T7161, Dl1l8H, and S982A revealed that they share the same
forward primer which contains ACC TCA AGG TAT TGG GAA CCT sequence
(3). Among these mutations, N501Y is the most frequent with 0.02% frequency.
Therefore, the designed reverse primer contains the CCA ACC CAC TTA TGG
TGT TGG sequence, and it has been tested.
[0020] A new SARS-CoV2 mutant called L452R with a mutation in spike
sequence is rapidly spreading in Denmark and the United States since 2020, and
2021, respectively. The analysis of the receptor-binding domain (RBD) indicates
that some of the mutations can enhance the attachment of S protein to ACE2 and
subsequently, the infectivity of the SARS-CoV-2 would be increased.
[0021] Studies have shown that L452R mutation is one of the monoclonal
antibody-resistant mutations and is of great importance in vaccination and
treatment of the patients. Henceforth, in terms of resistance to antibodies,
infectivity, and transmission rate, this mutation is important for public health and
especially the vaccination of the population.
[0022] It is revealed that two different SARS-CoV-2 strains can combine
their genomes and produce a third mutant hybrid. This recombination was first
observed in the California strain of the virus and indicates the probability of the
initiation of a new phase of the pandemic. This hybrid was the result of the
recombination of a highly contagious strain called B.1.1.7 from the U.K. with the
B.1.429 strain originated from California which can cause a new outbreak in Los
Angeles or even other parts of the world. This characteristic is due to the resistance
of this strain to some of the antibodies.
[0023] Since the new variants of COVID-19 are rapidly spreading through the countries and according to the reports of the ministry of health of different countries, several cases have been diagnosed with these new variants; therefore, the next outbreak of coronavirus is imminent.
OBJECTIVES OF THE EMBODIMENTS
[0024] The first objective is to provide the primers and probes which are
designed based on the conserved sequence of the novel coronavirus (SARS-CoV
2), and have a high detection rate of the target gene fragment.
[0025] The main object of the embodiments herein is to provide an accurate
differential detection kit for different variants of SARS-CoV-2 in COVID-19
patients.
[0026] The purpose of the embodiments is to provide a kit to detect the
presence and type of virus variant.
[0027] Accurate determination of the mutant region of various variants of the
SARS-CoV2.
[0028] The specific purpose of these embodiments is to design and prepare
specific probes for mutant regions in the new variants of the SARS-CoV-2.
[0029] Another object of the embodiments herein is to provide suitable
primers for amplification of the mutant regions, in different variants of the virus genome, simultaneously.
[0030] Next object of the embodiments herein is to provide a multiplex
reaction to perform a Real-time PCR.
[0031] Another object of the embodiments herein is to provide usable primers
and probes set to make the kit. The important point that should be considered is
this kit should possess no cross-reactions among positive samples of Coronavirus
(NL63, HKU1, 229E, OC43), Influenza A virus, Influenza B virus, Respiratory
syncytial virus, Adenovirus, Parainfluenza virus, Klebsiella pneumoniae,
Streptococcus pneumoniae, Haemophilus influenza, Pseudomonas aeruginosa,
Legionella pneumophila, Pertussis, Staphylococcus aureus, Mycoplasma
pneumoniae, and Chlamydia pneumoniae.
SUMMARY
[0032] This invention relates to the preparation and design of a diagnostic kit
for accurate detection of various variants as well as the wild type of SARS-CoV-2.
[0033] This kit is designed to identify the current common strains of the
virus.
[0034] According to this design, the steps will be as follows:
1- Accurate identification of mutated regions in the virus genome;
2- Designing primers and probes for replication and detection of mutated regions;
3- Simultaneous use of these sequences in a Real-time PCR run;
4- Setting the temperature to perform simultaneously reactions related to the
genes of conserved regions such as RdRp, N, spike, and mutated regions
in spike such as N501Y, L452R, D614G, and P681H.
[0035] During the operation steps, depending on the conditions of the tests,
changes and adjustments will probably be made in the work steps.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0036] Reagent Preparation (to be performed in the Reagent Processing
Area):
The component must be taken out and thawed at room temperature until
equilibrated. The Lyophilized Enzyme Mix must be resuspended in 400 pL
Enzyme Mix Buffer. Add 500 pL RNase-free water and mix by gently pipetting up
and down. Avoid generating air bubbles. To prevent wastage of the lyophilized
powder (i.e., to avoid leaving material in the tube), wash the wall of the tube by
pipetting. The tube will then be placed aside for 30 min.
[0037] Reaction Mix Preparation:
The recommended sample volume used in the reaction is 5 pL or 10 pL. This
mixture has to be prepared in two separate tubes designated, for instance, as vial 1 and vial 2. Aliquot of 15 pL (or 10 pL, depending on the sample volume) of the above reaction will be mixed into the PCR plate of the chosen PCR platform in two wells (first vial in first well and second vial in second well for each sample).
[0038] Aliquot into wells according to the number of samples to be tested
include one well for the positive control and one well for the negative control.
Transfer the reaction mix to the Sample Processing Area.
[0039] Sample Adding (to be performed in the Sample Processing Area):
For the 5 pL sample: Add 5 pL of the following into the appropriate wells
according to plate setup: Sample(s), positive control, and negative control. For 10
pL sample: Add 10 pL of the following into the appropriate wells according to
plate setup: Samples consist of diluted positive control, and negative control. After
adding the samples, cover the lid immediately. Spin down briefly using a
centrifuge to remove air bubbles. Transfer the mixture to the amplification area.
[0040] PCR Amplification (to be performed in the amplification and analysis
area):
Place the tubes on the sample holder in the instrument. Set up the test panel
according to the positions of positive control, negative control, and RNA samples.
Select the detection channels as follows:
a) Select FAM (RdRp gene) and HEX (N gene), and cy5 (spike gene) channels to
detect SARS-CoV-2 RNA by using the first vial.
b) Select ROX (RNase P) channel to detect the internal control.
c) Select VIC (D614G), CY5 (L452R), FAM (N501Y), JOE (P681H) channels to
detect one or more mutants of spike gene by using the second vial.
[0041] Program setting:
1. Reverse Transcription: 50°C, 15 min, 1 cycle;
2. cDNA Initial Denaturation: 95°C, 3 min, 1 cycle;
3. Denaturation: 95°C, 15 sec, 45-50 cycles;
4. Annealing, Extension and Fluorescence measurement: 55°C, 10 sec;
5. Cooling: 25°C, 10 sec, 1 cycle.
[0042] Result Interpretation:
After the reaction is completed, the results are automatically saved and the
amplification curves of the detected target RNA and the internal control are
analyzed separately. According to the analysis, the amplification plot will adjust
the start value, end value, and threshold value of the baseline (operators can adjust
the values according to the actual situation. Start value can be set within 3~15, end
value can be set within 5~20; operators can adjust the amplification curve of
negative control to make it linear or below the threshold line). Click "Analyze" to
perform the analysis and the parameters should meet the requirements mentioned
above.
[0043] Quality Control:
Lastly, record the qualitative results in the Plate window. COVID-19 PCR
Negative Control: None of the FAM, HEX, and Internal Control (ROX) channels
have a Ct value or Ct > 40. COVID-19 PCR Positive Control: FAM, HEX and
Internal Control (ROX) channels Ct < 35. The above requirements must be met at
the same time in the same experiment. Otherwise, this experiment is invalid and
needs to be repeated. According to the study of the reference value, the Ct
reference value for the target gene detected by this kit is 40, and the Ct reference
value of internal control is 40.

Claims (1)

CLAIMS [0044] The main claim of the embodiment herein is that in addition to detecting a positive / negative result for COVID-19 disease, the design of specific probes and primers of spike gene in positive patients, make a Real-time PCR-based method to rapidly identify and diagnose mutant variants in patients, carriers, and people who are suspected of being infected with mutated variants of SARS-CoV-2 with high accuracy. [0045] The next claim of the embodiment is that method is based on the use of Real-time PCR kits to diagnose SARS-CoV-2 infections. The basis for the result in this test is the proliferation detection containing specific mutation regions. [0046] Another claim of this embodiment is that in this method, probes with different colors are used for different mutations. The proliferation of the target areas is a sign of mutation(s) and the non-proliferation indicates no mutation of the related gene or genomic region. [0047] The next claim of the embodiment is that the reactions can be performed in more than one tube or vessel if the probe can be designed for different areas. In order to validate the specificity of the primer and probe set specific for the mutant region of interest, a positive control is necessary. References 1- Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A., Butterfield, Y. S., ... & Roper, R. L. (2003). The genome sequence of the SARS-associated coronavirus. Science, 300(5624), 1399-1404. 2- AminJafari, A., & Ghasemi, S. (2020). The possible of immunotherapy for COVID-19: A systematic review. InternationalImmunopharmacology, 83, 106455. 3- Mousavizadeh, L., & Ghasemi, S. (2021). Genotype and phenotype of COVID-19: Their roles in pathogenesis. Journal of Microbiology, Immunology and Infection, 54(2), pp. 1 59-163. 4- Lopez-Rincon, A., Perez-Romero, C., Tonda, A., Mendoza-Maldonado, L., Claassen, E., Garssen, J., & Kraneveld, A. D. (2021). Design of Specific Primer Sets for the Detection of B. 1.1. 7, B. 1.351, P. 1, B. 1.617. 2 and B.
1.1. 519 Variants of SARS-CoV-2 using Artificial Intelligence. bioRxiv.
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WO2023119307A1 (en) * 2021-12-23 2023-06-29 Krishnamurthy Girish Primer and probes for detection of omicron variant of sars-cov-2, methods and uses thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113461813A (en) * 2021-09-03 2021-10-01 上海良润生物医药科技有限公司 Paired antibody for detecting new coronavirus and application thereof
CN113461813B (en) * 2021-09-03 2021-12-14 上海良润生物医药科技有限公司 Paired antibody for detecting new coronavirus and application thereof
WO2023119307A1 (en) * 2021-12-23 2023-06-29 Krishnamurthy Girish Primer and probes for detection of omicron variant of sars-cov-2, methods and uses thereof
CN114369688A (en) * 2022-03-22 2022-04-19 深圳联合医学科技有限公司 Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte
CN114369688B (en) * 2022-03-22 2022-06-03 深圳联合医学科技有限公司 Compositions, kits, methods and uses for detecting a variant of SARS-CoV-2 Oncuronte

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