WO2022240381A1 - A method and kit for quantitative detection of sars-cov-2 by real-time pcr - Google Patents
A method and kit for quantitative detection of sars-cov-2 by real-time pcr Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
Definitions
- the invention relates to a method for quantitative detection of SARS-CoV-2 and a test kit in which said method is used.
- SARS-CoV-2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to a virus family called Coronaviridae and is a positive-sense single-stranded RNA virus.
- the RNA sequence is approximately 30.000-base length.
- SARS-CoV-2 has four structural proteins known as S (Spike), E (Envelope), M (Membrane), and N (Nucleocapsid) proteins.
- S Spike
- E envelope
- M Membrane
- N Nucleocapsid proteins.
- the N protein retains the RNA genome, and the S, E, and M proteins together form the viral envelope (1 ).
- the genome is known to have 6-11 functional open reading frames (ORFs) coding 27 proteins.
- ORFs functional open reading frames
- the ORF 1 a and ORF 1 b regions constitute approximately two-thirds of the genome length and encode a total of 16 non- structural proteins (nsps).
- the remaining one-third of the genome encodes 4 structural proteins (S, M, E, N) and at least 6 accessory proteins (2).
- PCR polymerase chain reaction
- immunoassays are used in diagnosis of SARS-CoV-2.
- the studies are carried out on samples obtained from blood, nose and throat cultures, urine, feces and various tissues taken from subjects.
- Reverse transcriptase PCR test on these gene regions, antigen test carried out with the monoclonal or polyclonal antibodies, various serological tests and the immunofluorescence tests are the basic tests used for diagnosis in laboratory (3).
- Russian patent application no. RU2731390C1 discloses a fast and reliable test for SARS-CoV-2 virus, based on cDNA synthesis using reverse transcription and classical real-time PCR.
- the detection of the highest number of cases in the shortest time and the determination of the viral relevance level of the population on a large scale within the widest possible framework constitute the basis of the effective fight against the pandemic.
- the average time to get results is 45 minutes, or even 60 minutes or more.
- the present invention relates to a method for quantitative detection of the presence of SARS-CoV-2 and a test kit for use in said method, providing a quantitatively significant, definitive diagnosis.
- a dual primer set and a fluorogenic probe design are developed for use in quantitatively detecting the presence of SARS-CoV-2.
- the molecular diagnosis of SARS-CoV-2 RNA genetic material is achieved in a quick, sensitive, specific and quantitative manner.
- the developed method, primer set and the fluorogenic probe designed within the context of the present invention enables quantitative detection of the presence of SARS-CoV-2 virus; and thus provides quickness, ease of use and cost reduction in detection of SARS-CoV-2 virus carriers in clinics.
- the detection kit according to the present invention has a high clinical sensitivity and is easy to use.
- the primer set and fluorogenic probe designed within the scope of the present invention have high sensitivity and specificity. Reliable data required for the definitive diagnosis of the presence of SARS-CoV-2 in an individual have been obtained at a high rate in all broad-spectrum sample applications.
- the average durationfor obtaining results is 33 minutes.
- the present invention makes it possible to test a higher number of individuals per unit time.
- RFU relative fluorescence unit
- the test becomes more sensitive and specific at the molecular level in comparison with the real-time PCR-based diagnosis or detection criteria and standard technical applications, thus increasing the reliability of the test results.
- the present invention provides SARS-CoV-2 RNA extracted from the biological sample to be quantitatively detected in line with its presence in the sample by the real-time RT-PCR method by using the dual primer set and fluorogenic probe designs which specifically bind to the nucleocapsid (N) gene region.
- the detection kit is resistant to the mutation effect since the target gene region determined for the real-time RT-PCR is the nucleocapsid (N), and the N gene region is a structurally more protected region against the possible mutations than other SARS-CoV-2 viral gene regions.
- N nucleocapsid
- the present invention does not pose any risk of infection.
- the present invention allows the biological sample to be realized without the need for any extraction procedure.
- a simpler, easier, low-cost, and quick method and a detection kit with a high analytical sensitivity have been developed, without occurrence of a chemical damage on the genomic material.
- the invention has the potential to be used in many different fields. It can be used in research of scientists working in the field of Molecular Biology, as a molecular in vitro diagnostic test in clinical laboratories and hospitals, or in monitoring the response to the treatment of the disease.
- the SARS-CoV-2 test kit for the real-time RT-PCR includes a first mixture comprising a primer that comprises a first forward primer (SEQ ID NO: 1) of the 5’- GGGAGCCTTGAATACACCAAAA-3’ structure corresponding to the positions 28.320 to 28.376 in the N gene region of SARS-CoV-2 RNA, and a first reverse primer (SEQ ID NO: 2) of the 5’- TGTAGCACGATTGCAGCATTG-3’ structure.
- said first mixture includes a first fluorogenic probe that comprises 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure.
- said first mixture further comprises a second forward primer (SEQ ID NO: 4) comprising 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5) 4 comprising 5’-GAGCGGCTGTCTCCACAAGT-3’ structure, and a second fluorogenic probe (SEQ ID NO: 6) comprising 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure.
- An exemplary embodiment of the test kit according to the invention further includes a second mixture that comprises a reagents mixture comprising one or more buffers, one or more cofactors, a plurality of nucleotides, and one or more enzymes.
- the second mixture is a reagents mixture comprising one or more buffers, Mg 2+ as a cofactor, deoxyribonucleotide triphosphate as nucleotides, one or more ribonuclease inhibitors, one or more reverse transcriptases, and one or more Taq polymerases.
- the method according the invention for quantitatively detecting the presence of SARS-CoV-2 in a biological sample includes detecting the presence of SARS-CoV-2 RNA in a reaction mixture that is obtained from a biological sample, by quantitative real-time PCR with the following steps: a) amplification of cDNA that corresponds to SARS-CoV-2 RNA, with a first forward primer (SEQ
- step (a) simultaneously with step (a), detection of amplification amount of cDNA by using a first fluorogenic probe (SEQ ID NO: 3) having 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure.
- a possible implementation of the method includes the comparison of the amplification amount of cDNA with that of at least one control sample.
- a preferred implementation of the method includes subjecting the reaction mixture and the biological sample to a mixing process prior to the step (a) without implementation of any RNA extraction procedure.
- Said mixing process can be performed using a vortex device.
- a preferred implementation of the method according to the invention includes obtaining said reaction mixture by mixing of a first mixture that comprises a first forward primer (SEQ ID NO: 1) of the 5’- GGGAGCCTTGAATACACCAAAA-3’ structure corresponding to the positions 28.320 to 28.376 in the N gene region of SARS-CoV-2 RNA, a first reverse primer (SEQ ID NO: 2) of the 5’-
- TGTAGCACGATTGCAGCATTG-3 structure, a second mixture comprising a mixture of the reagents comprising one or more buffers, one or more cofactors, more than one nucleotide, and one or more enzymes, and said biological sample with each other.
- the method comprises preferably obtaining said reaction mixture by mixing a first mixture comprising a primer set comprising a first forward primer (SEQ ID NO: 1) of the 5’-
- An exemplary embodiment of the method comprises preferably adding a second forward primer (SEQ ID NO: 4) comprising 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5) 5 comprising 5’-GAGCGGCTGTCTCCACAAGT-3’ structure, and a second fluorogenic probe (SEQ ID NO: 6) comprising 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure to the reaction mixture.
- a second forward primer SEQ ID NO: 4
- SEQ ID NO: 5 comprising 5’-GAGCGGCTGTCTCCACAAGT-3’ structure
- a second fluorogenic probe SEQ ID NO: 6 comprising 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure
- a preferred embodiment of the method comprises subjecting the reaction mixture to the following temperature protocol in the real-time RT-PCR test: i. keeping the reaction mixture at 50°C to 60°C (preferably, at 50°C) for at least 5 minutes (preferably, for 5 minutes), ii. keeping the reaction mixture at 95°C for 10 to 20 seconds (preferably, for 10 seconds) after step i, iii. subjecting the reaction mixture to the following temperature cycles 35 times after step ii: 95°C for 1 to 5 seconds (preferably, for 1 second) and then 60°C to 65°C (preferably, 60°C) for 1 to 5 seconds (preferably, 5 seconds).
- Fig. 1 shows an amplification diagram obtained from a fluorescent channel (FAM) of the negative samples in the exemplary study using the test kit according to the present invention.
- the abscissa axis represents the number of progressing cycles; the ordinate axis represents the relative fluorescence unit (RFU) value detected in each cycle.
- Fig. 2 shows an amplification diagram obtained from a fluorescent channel (HEX) of the positive samples in the exemplary study using the test kit according to the present invention.
- the abscissa axis represents the number of progressing cycles; the ordinate axis represents the relative fluorescence unit (RFU) value detected in each cycle.
- REU relative fluorescence unit
- Fig. 3 shows the specificity and sensitivity performances of the kit according to the present invention and of three separate commercially available dual kits (Kit-A, Kit-B and Kit-C) for the comparison in three separate column pairs starting from the left, respectively.
- the kit within the scope of the present invention is symbolized as “Kit*” and the specificity and sensitivity performance of said kit are shown with the rightmost column pair.
- the black column in the column pairs specific to each kit indicates the "specificity" percentage and the white column indicates the "sensitivity" percentage.
- the present invention relates to a method for the quantitative detection of the presence of SARS-CoV-2, and a test kit using this method for determining viral load.
- the present invention further relates to a method for quantitatively detecting the presence of SARS-CoV-2 in a biological sample, wherein said method comprises the following steps:
- step (a) simultaneously with step (a), detection of an amplification amount of the cDNA in the presence of a fluorogenic probe (SEQ ID NO: 3) with 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure, and comparison of said amount with at least one control sample.
- a fluorogenic probe SEQ ID NO: 3
- sample corresponds to biological samples which means any of blood, nasal culture, throat culture, urine, feces, a variety of cells, tissues and organs.
- the sample may be, for example, one or more samples selected from the upper respiratory tract samples (nasopharyngeal- oropharyngeal swab) and the lower respiratory tract samples (tracheal aspirate, sputum, bronchoalveolar lavage).
- the sample may be obtained in any manner known to a person skilled in the relevant art.
- the sample may contain one or more cell types, tissue or organs, and the sample can be taken from any part of the subject.
- the sample may be fresh, frozen, or stored at a temperature within the range between 2°C and 8°C. The duration of said storage can be, for example, up to 72 hours.
- control sample as used in the present specification can refer to one or both of the negative and positive control samples.
- positive control sample is used for the reagent stability control
- negative control sample is used for the contamination control.
- the control sample can be any of internal or external control samples. Internal control relates to housekeeping genes in the same PCR setup.
- RNA can be extracted from the biological sample by any RNA extraction method known in the art.
- RNA extraction methods include the use of commercial RNA extraction kits or viral nucleic acid buffer (VNAT), or lysis-based chemical methods.
- VNAT viral nucleic acid buffer
- the use of commercial RNA extraction kits or viral nucleic acid buffer (VNAT), or any of the lysis-based chemical methods are not applied for RNA extraction. Therefore, in a preferred implementation of the method according to the present invention, the biological sample is realized without any RNA extraction procedure, since such procedure is not necessary.
- a biological sample taken from a subject is provided in a sample container (for example, a sterile sample tube that contains 3 to 5 milliliters of a liquid viral transport medium (VTM)).
- VTM is a generally isotonic liquid broth or medium that is used for transferring a biological sample to a test. Such medium allows even sensitive organisms to survive at room temperature for relatively long periods.
- VTM examples are commercially available on the market, which may include, for example, amino acids, antimicrobial agents, buffer solution, salts, bovine serum albumin (BSA).
- the sample in the VTM can be mixed for homogenisation.
- the mixing process can be applied at relatively low speeds and for short periods of time in order to minimize denatu ration or fragmentation of genetic materials of cells and/or viruses in the sample.
- the mixing process can be implemented by treating the sample container using a centrifuge device or a vortex device, preferably in a vortex device. 7
- the real-time RT-PCR protocol implemented in the method according to the present invention is directed to a conversion of the viral RNA into a complementary DNA (cDNA) with reverse transcriptase enzyme, and then, an amplification of the cDNA in a polymerase chain reaction (PCR) in three steps.
- cDNA complementary DNA
- PCR polymerase chain reaction
- An exemplary real-time RT-PCR protocol analyzer implemented within the scope of the method according to the present invention can be adjusted in accordance with the temperature program shown in Table-4.
- a (dual) primer set that comprises a forward primer (SEQ ID NO: 1 ) consisting of nucleic acid sequences (oligonucleotide), and a reverse primer (SEQ ID NO: 2).
- Said primers are a dual primer set which specifically binds to the nucleocapsid (N) gene region of SARS-CoV-2 RNA.
- the determination of the amplification amount of the cDNA is performed using a fluorogenic probe (SEQ ID NO: 3) consisting of a nucleic acid sequence (oligonucleotide).
- Said fluorogenic probe includes a fluorescent (reporter) dye at 5' terminus and a quencher dye at 3' terminus. The probe binds to the cDNA between the forward and reverse primers. While the fluorescent dye and the quencher dye are attached to the probe, the quencher dye absorbs the light emitted by the fluorescent dye.
- the fluorescent dye can be any of the fluorescent dyes consisting of the group FAM (518 nm), TET (538 nm), JOE (548 nm), VIC (552 nm), HEX (553 nm), NED (575 nm), ROX (607 nm), Texas Red (615 nm), the Cy5 (667 nm) and emitting the different colors according to their maximum emission wavelengths.
- the fluorescent dye is HEX (553 nm).
- the quencher dye is any of TAMRA (tetramethylrhodamine) and dark quencher dyes.
- the fluorogenic probe generates a signal with a fluorescent radiation during cycles, allowing the amount of the RNA replicates to be measured.
- the emission amount of the fluorescent radiation provides real-time information about the amount of the target genomic material (i.e., SARS-CoV-2) and whether it is obtained or not.
- the method developed in accordance with the present invention has been observed to be sensitive enough to detect RNA replicates even in 1 milliliter or smaller samples (e.g., 100 microliters).
- the invention also relates to a detection kit for identifying a subject who is COVID-19 positive by quantitatively detecting the presence of SARS-CoV-2 in a biological sample.
- Said detection kit comprises a first mixture (Mixture-1 ) that comprises the above-described set of the forward primer (SEQ ID NO: 1 ) and reverse primer (SEQ ID NO: 2) and the fluorogenic probe for the detection of the presence of SARS-CoV-2 RNA in a supplied biological sample by quantitative real-time PCR as mentioned above.
- the primers mentioned herein i.e., forward primer and reverse primer
- the detection kit comprises a second mixture (Mixture-2) with a content that is suitable for running the real-time PCR process.
- the content in said second mixture may be a mixture of reagents including a buffer, a cofactor, nucleotides and enzymes.
- the second mixture can preferably comprise reverse transcriptase, Taq polymerase, nucleotides (dNTPs), cofactor (e.g., magnesium (Mg +2 )), ribonuclease inhibitor, and a buffer.
- the detection kit can comprise a positive control and a negative control as a control sample.
- the first mixture can comprise a set of a forward primer (SEQ ID NO: 1 ) and a reverse primer (SEQ ID NO: 2) and a fluorogenic probe (SEQ ID NO: 3), targeting the nucleocapsid (N) gene which encodes the outer sheath of the possible SARS-CoV-2 virus in the samples taken by the real-time PCR technique; along with a further set of a second forward primer (SEQ ID NO: 4) with 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5) with 5’- GAGCGGCTGTCTCCACAAGT-3’ structure and a second fluorogenic probe (SEQ ID NO: 6) with 5’- TTCTGACCTGAAGGCTCTGCGCG-3’ structure, targeting the RNAseP gene in human epithelial cell genome for control.
- a forward primer SEQ ID NO: 1
- a reverse primer SEQ ID NO: 2
- a fluorogenic probe SEQ ID NO: 3
- the second mixture can comprise a reverse transcriptase, a Taq polymerase, nucleotides (dNTPs; deoxyribonucleotide triphosphate), magnesium as a cofactor (Mg +2 ), a ribonuclease inhibitor and a buffer.
- dNTPs nucleotides
- Mg +2 magnesium as a cofactor
- An exemplary use of the detection kit according to the present invention includes preparation of a reaction mixture by mixing of the Mixture-1 and Mixture-2 with the biological sample, and application of the real-time RT-PCR with a temperature cycle in which the time periods and temperatures proposed in the present specification within the scope of the present invention are implemented.
- a normalization is performed to the genes with suitable reference genes.
- the normalization improves the reliability of the RT-PCR analysis and reveals any changes in the efficiency of the isolation, reverse- transcription and amplification steps; thus allowing comparisons between mRNA concentrations of different samples.
- reference genes housekeeping genes which are not tissue -specific but can be expressed in all cells can preferably be used in the diagnostic kit and method according to the present invention.
- Said reference gene may be the RNAseP gene in the human epithelial cell genome.
- the first mixture preferably comprises a primer set targeting the RnaseP gene and a fluorogenic probe as an internal control.
- the fluorogenic probe specific for said RNAseP gene comprises a fluorescent dye at its 5' terminus and a quencher dye at its 3' terminus; the dyes are preferably FAM (fluorescent dye) and BHQ1 (quencher dye).
- a preferred embodiment of the diagnostic kit according to the present invention may comprise either or both of a positive control and a negative control as an external control sample.
- the negative control can be RNase-free water
- the positive control can be a mixture of a synthetic SARS-CoV-2 gene and RNAseP gene.
- RNA samples were obtained and said RNA samples were then stored at a temperature within the range between 2°C and 8°C (4°C in this exemplary experiment).
- RNA extraction with the Viral Nucleic Acid Buffer can be performed by using costly, time-consuming known techniques, yet that not essential in the method according to the present invention.
- the viral material and patient’s genomic material in the fluid sample can be obtained preferably by homogenizing the RNA sample by vortexing, without necessitating an extra degradation or extraction procedure in the VTM.. Thanks to the sensitivity of the kit according to the present invention, diagnosis can be made and results can be obtained even from small amounts of RNA which have already come out of the envelope in said material.
- RNA samples are subjected to a vortexing process (e.g., for 5 to 10 seconds). Then, a PCR reaction mixture was obtained by combining the RNA samples with the diagnostic kit according to the present invention.
- the reaction mixture was dispensed into plates (e.g., a 96-well plate) or strips that are prepared in accordance with the number of the sample tubes, for example 8 pi per well (e.g., 4 microliters of the Mixture 1 and 4 microliters of the Mixture 2).
- 10 sample was added onto the PCR reaction mixtures in such a way that, e.g., 4 microliters of the biological sample were poured into each of the respective wells.
- Mixture 1 A mixture that comprises the forward and reverse primers for the N and
- RNase-P genes and a fluorescently labeled fluorogenic probe (e.g., Taqman probe).
- a fluorescently labeled fluorogenic probe e.g., Taqman probe
- Mixture 2 A mixture that comprises Reverse Transcriptase and Taq Polymerase enzymes, nucleotides (dNTPs); one or more buffers that contain magnesium (Mg +2 ) as a cofactor and one or more ribonuclease inhibitors.
- each comparative reaction mixture 32, 10 and 10 microliters of the respective first mixtures thereof were added, respectively; 3, 5 and 5 microliters of the second respective mixtures thereof were added, respectively; and 15, 5 and 5 microliters of the RNA samples were added, respectively.
- the total volumes of the resulting reaction mixtures were 50, 20 and 20 microliters, respectively.
- Kit-A and the content of Kit-B are indicated in the Table 1 and Table 2 below, respectively.
- Kit-C Content for the Kit-C for 100 tests is indicated as follows: 1000 microliters of RT-PCR 2X MasterMix, 500 microliters of S-gene/GAPDH Mix and 20 microliters of S-gene/GAPDH Positive Control.
- the protocol applied with the kit according to the present invention is directed to conversion of the viral RNA into complementary DNA (cDNA) with the reverse transcriptase enzyme, followed by amplification of the cDNA in a polymerase chain reaction (PCR) in three steps.
- cDNA complementary DNA
- PCR polymerase chain reaction
- the analyzer is set according to the temperature program shown in the Table 4.
- the reaction mixtures in the plates or strips that are placed in the analyzer are: first, kept at 50°C to 60°C (here, at 50°C) for at least 5 minutes (here, 5 minutes), then, kept at 95°C for 10 to 20 seconds (here, 10 seconds), then, exposed to the following temperature cycle for 35 times: o 95°C for 1 to 5 seconds (here, 1 seconds) and o 60°C to 65°C (here, 60°C) for 1 to 5 seconds (here, 5 seconds).
- the total application time of the temperature program (result time) (including the dwell times for device-based cycle transition temperature changes, in accordance with the Biorad CFX-96 Touch analyzer operated by Bio-Rad CFX Maestro Software) was 33 minutes in gross; the net total time (reaction time) of the temperature program was determined as 8.67 minutes.
- the processes in which the reaction mixture temperature is kept at 95°C in the cycles are directed to cDNA denaturation, thereby obtaining denatured cDNA strands (thus, RNA replicates).
- the processes in which the temperature is kept at 60°C are directed to the primer and probe binding to the denatured cDNA strands, and to the amplification by DNA polymerase.
- the cDNA amplified in these temperature cycles creates a higher number of RNA replicates with each cycle.
- the probe generates a signal with a fluorescent radiation during the cycles, allowing the amount of the RNA replicates to be measured.
- the emission amount of the fluorescent radiation provides real-time information about the presence and amount of the target genomic material (i.e., SARS-CoV-2) obtained.
- the total application times of the temperature program to the respective reaction mixtures for Kit-A, Kit-B and Kit-C are 80, 65 and 60 minutes, respectively; and the temperature programs for each of these are respectively presented in Table 5, Table 6 and Table 7 below.
- GP, YN, GN and YP correspond to the true positive, false negative, true negative and false positive, respectively.
- the kit according to the present invention detected all of 384 positives as positive, while it detected all of 400 negatives as negative.
- the calculated sensitivity and specificity values are presented in the Table 5 below. Table 5. Sensitivity and specificity values obtained with the kit according to the present invention
- Kit-A The sensitivity obtained with Kit-A was determined as 95.88% with a confidence interval of 89.78% to 98.87%; the specificity was determined as 93.20% with a confidence interval of 86.5% to 97.72%.
- the 14 number of true positives was 93, the number of false positives was 7, the number of true negatives was 96, and the number of false negatives was 4.
- Kit- B The sensitivity obtained with Kit- B was determined as 96.94% with a confidence interval of 91 .31 % to 99.36%; the specificity was determined as 95.10% with a confidence interval of 88.93% to 98.39%.
- the number of true positives was 95, the number of false positives was 5, the number of true negatives was 97, and the number of false negatives was 3.
- Kit- C The sensitivity obtained with Kit- C was determined as 94.95% with a confidence interval of 88.61 % to 98.34%; the specificity was determined as 94.06% with a confidence interval of 87.52% to 97.79%.
- the number of true positives was 94, the number of false positives was 6, the number of true negatives was 97, and the number of false negatives was 3.
- the extraction step which is normally applied to the sample as a preliminary step in the detection of SARS- CoV-2 by real-time PCR during the pandemic period has some difficulties such as the supply of extraction kits, the additional cost that is brought by said kits, long extraction times, lack of a uniform extraction procedure, experience requirementin relation with the respective procedure, and chemical damage to the genomic material caused by lysis-based chemical methods. Due to such difficulties in the extraction process, a sample can be subjected to RT-PCR procedure as a simple, easy, cost-effective and quick solution, without any pretreatment for extraction. However, in this case, inhibition may be observed in some amplification systems and/or analytical sensitivity of the test may change, since the relevant pretreatment step is omitted from the respective method. Thanks to the kit according to the present invention, a rapid, highly accurate and cost-effective SARS-CoV-2 diagnosis is rendered possible even when an RNA extraction procedure is not performed.
Abstract
The present invention proposes a SARS-CoV-2 test kit for real-time RT-PCR. Said test kit includes a primer set comprising a first forward primer (SEQ ID NO: 1) and a first reverse primer (SEQ ID NO: 2), and preferably, also a first mixture comprising a second fluorogenic probe (SEQ ID NO: 3) as a probe. A preferred embodiment of the invention further includes a second mixture comprising a reagents mixture that comprises one or more buffers, one or more cofactors, more than one nucleotides, and one or more enzymes. The present invention further proposes a real-time RT-PCR test performed by using said test kit.
Description
1
A METHOD AND KIT FOR QUANTITATIVE DETECTION OF SARS-CoV-2 BY REAL-TIME PCR
Technical Field
The invention relates to a method for quantitative detection of SARS-CoV-2 and a test kit in which said method is used.
Prior Art
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to a virus family called Coronaviridae and is a positive-sense single-stranded RNA virus. The RNA sequence is approximately 30.000-base length. Like other coronaviruses, SARS-CoV-2 has four structural proteins known as S (Spike), E (Envelope), M (Membrane), and N (Nucleocapsid) proteins. The N protein retains the RNA genome, and the S, E, and M proteins together form the viral envelope (1 ).
The genome is known to have 6-11 functional open reading frames (ORFs) coding 27 proteins. The ORF 1 a and ORF 1 b regions constitute approximately two-thirds of the genome length and encode a total of 16 non- structural proteins (nsps). The remaining one-third of the genome encodes 4 structural proteins (S, M, E, N) and at least 6 accessory proteins (2).
In general, the polymerase chain reaction (in short: PCR) studies and immunoassays are used in diagnosis of SARS-CoV-2. The studies are carried out on samples obtained from blood, nose and throat cultures, urine, feces and various tissues taken from subjects. Reverse transcriptase PCR test on these gene regions, antigen test carried out with the monoclonal or polyclonal antibodies, various serological tests and the immunofluorescence tests are the basic tests used for diagnosis in laboratory (3).
Today, there are studies for performing quantitative and qualitative detection of SARS-COV-2 using real time PCR (RT-PCR) technique in terms of sensitivity. These tests are obtained by designing various probes and primers with the RT-PCR protocols known in the art. Among these studies, there are the real-time PCR test kits targeting various regions of the nucleocapsid (N) gene in the SARS-CoV-2 genome (4, 5, 6).
Due to the rapid spread of SARS-CoV-2 on a global scale, very high genomic diversity is observed in the gene position and nucleotide sequences. The evolution of SARS-CoV-2 at molecular level and the resulting genetic diversity are prominent problems from which the molecular diagnostic kit manufacturers currently suffer. Therefore, the manufacturers need to regularly update and optimize the oligonucleotide sequences through frequent analyzes of updated genomic sequences to increase their accuracy and reliability. The ORF1 b and nucleocapsid (N) gene regions in the SARS-CoV-2 genome are primarily preferred in clinical diagnostic tests since they are structurally protected against mutations (3, 8).
In the RT-PCR and reverse transcriptase PCR-based experiments on sensitivity of different regions (ORF1 ab, E, N, RdRp, S) of the SARS-CoV-2 genome during virus detection stage, it was determined that
2 the N gene has the highest (87%) analytical sensitivity. The ORFl ab gene region is ranked second with a sensitivity of 82% (7).
In the state of the art, Russian patent application no. RU2731390C1 discloses a fast and reliable test for SARS-CoV-2 virus, based on cDNA synthesis using reverse transcription and classical real-time PCR.
Molecular analyzes are still being developed to optimize the clinical sensitivity and ease of use of present SARS-CoV-2 diagnostic kits. Molecular tests may not be able to diagnose COVID-19 if they have low sensitivity. Although multiple factors affect the test results, the technical shortcomings in the product design such as unstable reagents, inappropriate amounts, irrational oligonucleotide design should be avoided. In addition, some primers and probe designs are inherently more sensitive than others. Therefore, the most important problem in currently used different diagnostic kits is the problem in gathering necessary data for clearly making a definite diagnosis in subjects with different-spectrum viral loads.
Furthermore, the detection of the highest number of cases in the shortest time and the determination of the viral relevance level of the population on a large scale within the widest possible framework constitute the basis of the effective fight against the pandemic. According to the standard technical application protocols, the average time to get results is 45 minutes, or even 60 minutes or more.
Summary of the Invention
The present invention relates to a method for quantitative detection of the presence of SARS-CoV-2 and a test kit for use in said method, providing a quantitatively significant, definitive diagnosis.
Primary object of the present invention is to eliminate the shortcomings encountered in the prior art. Another object of the present invention is to provide a method for quantitatively detecting the presence of SARS- CoV-2 virus in a biological sample. Another object of the present invention is to provide a detection kit for detecting an individual who is COVID-19 positive, i.e., that carries said virus, by quantitatively detecting the presence of SARS-CoV-2 virus in a biological sample.
In accordance with the present invention, a dual primer set and a fluorogenic probe design are developed for use in quantitatively detecting the presence of SARS-CoV-2. With the present invention, the molecular diagnosis of SARS-CoV-2 RNA genetic material is achieved in a quick, sensitive, specific and quantitative manner. The developed method, primer set and the fluorogenic probe designed within the context of the present invention, enables quantitative detection of the presence of SARS-CoV-2 virus; and thus provides quickness, ease of use and cost reduction in detection of SARS-CoV-2 virus carriers in clinics. The detection kit according to the present invention has a high clinical sensitivity and is easy to use.
The primer set and fluorogenic probe designed within the scope of the present invention have high sensitivity and specificity. Reliable data required for the definitive diagnosis of the presence of SARS-CoV-2 in an individual have been obtained at a high rate in all broad-spectrum sample applications.
3
In an exemplary application protocol within the scope of the present invention, with standard devices that are used, the average durationfor obtaining results is 33 minutes. The present invention makes it possible to test a higher number of individuals per unit time. When the signal analysis values of the relative fluorescence unit (abbreviated as: RFU) of the reactions performed with the time criteria in the developed application protocol were examined, no decrease in the signal quality or intensity was observed. Thus, the unit reaction time was shortened, and it was possible to analyze the samples taken from more subjects in a unit time.
In the tests performed with the detection kit according to the present invention, the test becomes more sensitive and specific at the molecular level in comparison with the real-time PCR-based diagnosis or detection criteria and standard technical applications, thus increasing the reliability of the test results. In obtaining these results, the high specificity of the primer and probe oligonucleotide sequence designs developed specific to the viral RNA, and the optimization of the product content that include the enzyme contents used for the amplification steps and all other components, in the combinations which will yield the optimum results, are also effective.
The present invention provides SARS-CoV-2 RNA extracted from the biological sample to be quantitatively detected in line with its presence in the sample by the real-time RT-PCR method by using the dual primer set and fluorogenic probe designs which specifically bind to the nucleocapsid (N) gene region.
In the present invention, the detection kit is resistant to the mutation effect since the target gene region determined for the real-time RT-PCR is the nucleocapsid (N), and the N gene region is a structurally more protected region against the possible mutations than other SARS-CoV-2 viral gene regions. Thus, an important advantage has been provided in the diagnosis against possible mutant virus strains.
The present invention does not pose any risk of infection.
The present invention allows the biological sample to be realized without the need for any extraction procedure. Thus, a simpler, easier, low-cost, and quick method and a detection kit with a high analytical sensitivity have been developed, without occurrence of a chemical damage on the genomic material.
The invention has the potential to be used in many different fields. It can be used in research of scientists working in the field of Molecular Biology, as a molecular in vitro diagnostic test in clinical laboratories and hospitals, or in monitoring the response to the treatment of the disease.
The SARS-CoV-2 test kit for the real-time RT-PCR according to the invention includes a first mixture comprising a primer that comprises a first forward primer (SEQ ID NO: 1) of the 5’- GGGAGCCTTGAATACACCAAAA-3’ structure corresponding to the positions 28.320 to 28.376 in the N gene region of SARS-CoV-2 RNA, and a first reverse primer (SEQ ID NO: 2) of the 5’- TGTAGCACGATTGCAGCATTG-3’ structure. In a possible embodiment of the invention, said first mixture includes a first fluorogenic probe that comprises 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure. In a possible embodiment of the invention, said first mixture further comprises a second forward primer (SEQ ID NO: 4) comprising 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5)
4 comprising 5’-GAGCGGCTGTCTCCACAAGT-3’ structure, and a second fluorogenic probe (SEQ ID NO: 6) comprising 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure. An exemplary embodiment of the test kit according to the invention further includes a second mixture that comprises a reagents mixture comprising one or more buffers, one or more cofactors, a plurality of nucleotides, and one or more enzymes. In said embodiment, the second mixture is a reagents mixture comprising one or more buffers, Mg2+ as a cofactor, deoxyribonucleotide triphosphate as nucleotides, one or more ribonuclease inhibitors, one or more reverse transcriptases, and one or more Taq polymerases.
The method according the invention for quantitatively detecting the presence of SARS-CoV-2 in a biological sample includes detecting the presence of SARS-CoV-2 RNA in a reaction mixture that is obtained from a biological sample, by quantitative real-time PCR with the following steps: a) amplification of cDNA that corresponds to SARS-CoV-2 RNA, with a first forward primer (SEQ
ID NO: 1 ) having 5’-GGGAGCCTTGAATACACCAAAA-3’ structure and a first reverse primer (SEQ ID NO: 2) having 5’-TGTAGCACGATTGCAGCATTG-3’ structure, b) simultaneously with step (a), detection of amplification amount of cDNA by using a first fluorogenic probe (SEQ ID NO: 3) having 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure.
A possible implementation of the method includes the comparison of the amplification amount of cDNA with that of at least one control sample.
A preferred implementation of the method includes subjecting the reaction mixture and the biological sample to a mixing process prior to the step (a) without implementation of any RNA extraction procedure. Said mixing process can be performed using a vortex device.
A preferred implementation of the method according to the invention includes obtaining said reaction mixture by mixing of a first mixture that comprises a first forward primer (SEQ ID NO: 1) of the 5’- GGGAGCCTTGAATACACCAAAA-3’ structure corresponding to the positions 28.320 to 28.376 in the N gene region of SARS-CoV-2 RNA, a first reverse primer (SEQ ID NO: 2) of the 5’-
TGTAGCACGATTGCAGCATTG-3’ structure, a second mixture comprising a mixture of the reagents comprising one or more buffers, one or more cofactors, more than one nucleotide, and one or more enzymes, and said biological sample with each other.
For example, the method comprises preferably obtaining said reaction mixture by mixing a first mixture comprising a primer set comprising a first forward primer (SEQ ID NO: 1) of the 5’-
GGGAGCCTTGAATACACCAAAA-3’ structure and a first reverse primer (SEQ ID NO: 2) of the 5’- TGTAGCACGATTGCAGCATTG-3’ structure, a second mixture comprising a mixture of the reagents comprising one or more buffers, Mg2+ as a cofactor, more than one nucleotide, one or more ribonuclease inhibitors, one or more reverse transcriptases, and one or more Taq polymerases, and said biological sample with each other.
An exemplary embodiment of the method comprises preferably adding a second forward primer (SEQ ID NO: 4) comprising 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5)
5 comprising 5’-GAGCGGCTGTCTCCACAAGT-3’ structure, and a second fluorogenic probe (SEQ ID NO: 6) comprising 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure to the reaction mixture.
A preferred embodiment of the method comprises subjecting the reaction mixture to the following temperature protocol in the real-time RT-PCR test: i. keeping the reaction mixture at 50°C to 60°C (preferably, at 50°C) for at least 5 minutes (preferably, for 5 minutes), ii. keeping the reaction mixture at 95°C for 10 to 20 seconds (preferably, for 10 seconds) after step i, iii. subjecting the reaction mixture to the following temperature cycles 35 times after step ii: 95°C for 1 to 5 seconds (preferably, for 1 second) and then 60°C to 65°C (preferably, 60°C) for 1 to 5 seconds (preferably, 5 seconds).
Brief Description of the Figures
Fig. 1 shows an amplification diagram obtained from a fluorescent channel (FAM) of the negative samples in the exemplary study using the test kit according to the present invention. The abscissa axis represents the number of progressing cycles; the ordinate axis represents the relative fluorescence unit (RFU) value detected in each cycle.
Fig. 2 shows an amplification diagram obtained from a fluorescent channel (HEX) of the positive samples in the exemplary study using the test kit according to the present invention. The abscissa axis represents the number of progressing cycles; the ordinate axis represents the relative fluorescence unit (RFU) value detected in each cycle.
Fig. 3 shows the specificity and sensitivity performances of the kit according to the present invention and of three separate commercially available dual kits (Kit-A, Kit-B and Kit-C) for the comparison in three separate column pairs starting from the left, respectively. The kit within the scope of the present invention is symbolized as “Kit*” and the specificity and sensitivity performance of said kit are shown with the rightmost column pair. Here, the black column in the column pairs specific to each kit indicates the "specificity" percentage and the white column indicates the "sensitivity" percentage.
Detailed Description of the Invention
The present invention relates to a method for the quantitative detection of the presence of SARS-CoV-2, and a test kit using this method for determining viral load.
The present invention further relates to a method for quantitatively detecting the presence of SARS-CoV-2 in a biological sample, wherein said method comprises the following steps:
- provision of a biological sample, detection of the presence of SARS-CoV-2 RNA in said biological sample by quantitative real-time PCR with the following steps:
6 a) amplification of a cDNA that corresponds to SARS-CoV-2 RNA in the presence of a forward primer
(SEQ ID NO: 1 ) with 5’-GGGAGCCTTGAATACACCAAAA-3’ structure and a reverse primer (SEQ ID NO: 2) with 5’-TGTAGCACGATTGCAGCATTG-3’ structure, b) simultaneously with step (a), detection of an amplification amount of the cDNA in the presence of a fluorogenic probe (SEQ ID NO: 3) with 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure, and comparison of said amount with at least one control sample.
The term “sample” as used in the present specification corresponds to biological samples which means any of blood, nasal culture, throat culture, urine, feces, a variety of cells, tissues and organs. The sample may be, for example, one or more samples selected from the upper respiratory tract samples (nasopharyngeal- oropharyngeal swab) and the lower respiratory tract samples (tracheal aspirate, sputum, bronchoalveolar lavage). The sample may be obtained in any manner known to a person skilled in the relevant art. The sample may contain one or more cell types, tissue or organs, and the sample can be taken from any part of the subject. The sample may be fresh, frozen, or stored at a temperature within the range between 2°C and 8°C. The duration of said storage can be, for example, up to 72 hours.
The term “subject”, “individual” or “patient” as it is used in the present specification refers to any mammal. The term “control sample” as used in the present specification can refer to one or both of the negative and positive control samples. The term positive control sample is used for the reagent stability control, and the term negative control sample is used for the contamination control. The control sample can be any of internal or external control samples. Internal control relates to housekeeping genes in the same PCR setup.
In an implementation of the present invention, RNA can be extracted from the biological sample by any RNA extraction method known in the art. Examples to said RNA extraction methods include the use of commercial RNA extraction kits or viral nucleic acid buffer (VNAT), or lysis-based chemical methods. In a preferred implementation of the present invention, the use of commercial RNA extraction kits or viral nucleic acid buffer (VNAT), or any of the lysis-based chemical methods are not applied for RNA extraction. Therefore, in a preferred implementation of the method according to the present invention, the biological sample is realized without any RNA extraction procedure, since such procedure is not necessary.
In the invention which is the subject of the present application, at the step of provision of the biological sample, a biological sample taken from a subject is provided in a sample container (for example, a sterile sample tube that contains 3 to 5 milliliters of a liquid viral transport medium (VTM)). VTM is a generally isotonic liquid broth or medium that is used for transferring a biological sample to a test. Such medium allows even sensitive organisms to survive at room temperature for relatively long periods. Various VTM examples are commercially available on the market, which may include, for example, amino acids, antimicrobial agents, buffer solution, salts, bovine serum albumin (BSA).
Prior to proceeding to the next step in the method according to the present invention, the sample in the VTM can be mixed for homogenisation. The mixing process can be applied at relatively low speeds and for short periods of time in order to minimize denatu ration or fragmentation of genetic materials of cells and/or viruses in the sample. The mixing process can be implemented by treating the sample container using a centrifuge device or a vortex device, preferably in a vortex device.
7
In the present invention, quantitative detection of the presence of SARS-CoV-2 RNA in the biological sample is performed via real-time RT-PCR. The real-time RT-PCR protocol implemented in the method according to the present invention is directed to a conversion of the viral RNA into a complementary DNA (cDNA) with reverse transcriptase enzyme, and then, an amplification of the cDNA in a polymerase chain reaction (PCR) in three steps. An exemplary real-time RT-PCR protocol analyzer implemented within the scope of the method according to the present invention can be adjusted in accordance with the temperature program shown in Table-4.
In the method according to the present invention, in the step of amplification of the cDNA that corresponds to SARS-CoV-2 RNA, a (dual) primer set is used that comprises a forward primer (SEQ ID NO: 1 ) consisting of nucleic acid sequences (oligonucleotide), and a reverse primer (SEQ ID NO: 2). Said primers are a dual primer set which specifically binds to the nucleocapsid (N) gene region of SARS-CoV-2 RNA.
In the method according to the present invention, simultaneously with the binding of the primers to the relevant region and the amplification of the cDNA, the determination of the amplification amount of the cDNA is performedusing a fluorogenic probe (SEQ ID NO: 3) consisting of a nucleic acid sequence (oligonucleotide). Said fluorogenic probe includes a fluorescent (reporter) dye at 5' terminus and a quencher dye at 3' terminus. The probe binds to the cDNA between the forward and reverse primers. While the fluorescent dye and the quencher dye are attached to the probe, the quencher dye absorbs the light emitted by the fluorescent dye. In the extension phase of the PCR reaction, the probe is fragmented, the fluorescent dye is released, and in this way, the emitted irradiation is detected. The fluorescent dye can be any of the fluorescent dyes consisting of the group FAM (518 nm), TET (538 nm), JOE (548 nm), VIC (552 nm), HEX (553 nm), NED (575 nm), ROX (607 nm), Texas Red (615 nm), the Cy5 (667 nm) and emitting the different colors according to their maximum emission wavelengths. In a preferred implementation of the invention, the fluorescent dye is HEX (553 nm). The quencher dye is any of TAMRA (tetramethylrhodamine) and dark quencher dyes.
The fluorogenic probe generates a signal with a fluorescent radiation during cycles, allowing the amount of the RNA replicates to be measured. The emission amount of the fluorescent radiation provides real-time information about the amount of the target genomic material (i.e., SARS-CoV-2) and whether it is obtained or not. The method developed in accordance with the present invention has been observed to be sensitive enough to detect RNA replicates even in 1 milliliter or smaller samples (e.g., 100 microliters).
The invention also relates to a detection kit for identifying a subject who is COVID-19 positive by quantitatively detecting the presence of SARS-CoV-2 in a biological sample.
Said detection kit comprises a first mixture (Mixture-1 ) that comprises the above-described set of the forward primer (SEQ ID NO: 1 ) and reverse primer (SEQ ID NO: 2) and the fluorogenic probe for the detection of the presence of SARS-CoV-2 RNA in a supplied biological sample by quantitative real-time PCR as mentioned above. The primers mentioned herein (i.e., forward primer and reverse primer) correspond to the positions 28.320 to 28.376 in the N gene region of the SARS-CoV-2 RNA.
8
In a preferred embodiment of the invention, the detection kit comprises a second mixture (Mixture-2) with a content that is suitable for running the real-time PCR process. The content in said second mixture may be a mixture of reagents including a buffer, a cofactor, nucleotides and enzymes. For example, the second mixture can preferably comprise reverse transcriptase, Taq polymerase, nucleotides (dNTPs), cofactor (e.g., magnesium (Mg+2)), ribonuclease inhibitor, and a buffer.
In a preferred embodiment of the invention, the detection kit can comprise a positive control and a negative control as a control sample.
In a preferred embodiment of the invention, the first mixture can comprise a set of a forward primer (SEQ ID NO: 1 ) and a reverse primer (SEQ ID NO: 2) and a fluorogenic probe (SEQ ID NO: 3), targeting the nucleocapsid (N) gene which encodes the outer sheath of the possible SARS-CoV-2 virus in the samples taken by the real-time PCR technique; along with a further set of a second forward primer (SEQ ID NO: 4) with 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5) with 5’- GAGCGGCTGTCTCCACAAGT-3’ structure and a second fluorogenic probe (SEQ ID NO: 6) with 5’- TTCTGACCTGAAGGCTCTGCGCG-3’ structure, targeting the RNAseP gene in human epithelial cell genome for control.
In a preferred embodiment of the invention, the second mixture can comprise a reverse transcriptase, a Taq polymerase, nucleotides (dNTPs; deoxyribonucleotide triphosphate), magnesium as a cofactor (Mg+2), a ribonuclease inhibitor and a buffer.
An exemplary use of the detection kit according to the present invention includes preparation of a reaction mixture by mixing of the Mixture-1 and Mixture-2 with the biological sample, and application of the real-time RT-PCR with a temperature cycle in which the time periods and temperatures proposed in the present specification within the scope of the present invention are implemented.
In order to minimize possible problems which may occur in the analysis of the test results obtained by the use of the detection kit according to the present invention through real-time PCR method, preferably a normalization is performed to the genes with suitable reference genes. The normalization improves the reliability of the RT-PCR analysis and reveals any changes in the efficiency of the isolation, reverse- transcription and amplification steps; thus allowing comparisons between mRNA concentrations of different samples. Accordingly, reference genes (housekeeping genes) which are not tissue -specific but can be expressed in all cells can preferably be used in the diagnostic kit and method according to the present invention. Said reference gene may be the RNAseP gene in the human epithelial cell genome. Within the scope of the invention, the first mixture preferably comprises a primer set targeting the RnaseP gene and a fluorogenic probe as an internal control. The fluorogenic probe specific for said RNAseP gene comprises a fluorescent dye at its 5' terminus and a quencher dye at its 3' terminus; the dyes are preferably FAM (fluorescent dye) and BHQ1 (quencher dye).
9
A preferred embodiment of the diagnostic kit according to the present invention may comprise either or both of a positive control and a negative control as an external control sample. Here, the negative control can be RNase-free water, and the positive control can be a mixture of a synthetic SARS-CoV-2 gene and RNAseP gene.
Examples
It is possible to develop a wide variety of applications within the scope of the present invention that relates to the method and detection kit for quantitative detection of SARS-CoV-2 by the real-time RT-PCR; and the scope of the invention defined by the claims cannot be limited to the examples described herein.
A study was conducted for measuring the sensitivity and specificity of the kit according to the present invention, with 384 (i.e., 96x4) positive samples and 400 negative samples according to reference clinical data including different SARS-CoV-2 variants.
Example 1 - Sampling:
Upper respiratory tract samples (nasopharyngeal-oropharyngeal swab) and lower respiratory tract samples (e.g., tracheal aspirate, sputum, bronchoalveolar lavage) taken from individuals, for instance by means of dacron or polyester flocked swabs, without causing PCR inhibition, are transferred into sterile sample tubes that contain a liquid viral transport medium (Viral Transport Medium, VTM) with a volume within the range between 3 and 5 ml. Thus, RNA samples were obtained and said RNA samples were then stored at a temperature within the range between 2°C and 8°C (4°C in this exemplary experiment).
RNA extraction with the Viral Nucleic Acid Buffer (VNAT) can be performed by using costly, time-consuming known techniques, yet that not essential in the method according to the present invention. In terms of cost and time saving and in since it does not cause chemical damage to the genomic material, the viral material and patient’s genomic material in the fluid sample can be obtained preferably by homogenizing the RNA sample by vortexing, without necessitating an extra degradation or extraction procedure in the VTM.. Thanks to the sensitivity of the kit according to the present invention, diagnosis can be made and results can be obtained even from small amounts of RNA which have already come out of the envelope in said material.
In this way, even if a sample kept in the sample container within the above-mentioned storage temperature range, and analyzed even after days without degradation , no decrease in the analysis sensitivity is observed.
Example 2- Pre-RT-PCR application (reaction setup):
The RNA samples are subjected to a vortexing process (e.g., for 5 to 10 seconds). Then, a PCR reaction mixture was obtained by combining the RNA samples with the diagnostic kit according to the present invention. The reaction mixture was dispensed into plates (e.g., a 96-well plate) or strips that are prepared in accordance with the number of the sample tubes, for example 8 pi per well (e.g., 4 microliters of the Mixture 1 and 4 microliters of the Mixture 2). After the dispensing of Mixture 1 and Mixture 2 was completed,
10 sample was added onto the PCR reaction mixtures in such a way that, e.g., 4 microliters of the biological sample were poured into each of the respective wells. If a plate was used, seals of each of the respective wells were closed; and if a strip was used, seals of each of the wells were closed, and the reaction mixtures were subjected to a spinning process for, e.g., 5 to 10 seconds, and then the reaction mixture was placed into an analyzer (Biorad CFX-96 Touch operated by Bio-Rad CFX Maestro Software) for RT-qPCR.
The mixtures that are used in the exemplary studies and that are included in the exemplary kit within the scope of the present invention are as follows: - Mixture 1 : A mixture that comprises the forward and reverse primers for the N and
RNase-P genes and a fluorescently labeled fluorogenic probe (e.g., Taqman probe).
Mixture 2: A mixture that comprises Reverse Transcriptase and Taq Polymerase enzymes, nucleotides (dNTPs); one or more buffers that contain magnesium (Mg+2) as a cofactor and one or more ribonuclease inhibitors.
Comparative experiments were performed by using three separate commercially available dual kits (Bioeksen RT-QPCR KIT - LOT 2B00917E150R100-TK20; COYOTE DIRECT DETECT SARS COV-2 DETECTION KIT - 6018000902/06/08/CE SCOV2-0 V1.0; and DIAGEN REF NO: DIA-CV19-2; which are hereby named Kit-A, Kit-B, and Kit-C, respectively), each containing a respective first mixture and a respective second mixture. In the preparation of each comparative reaction mixture, 32, 10 and 10 microliters of the respective first mixtures thereof were added, respectively; 3, 5 and 5 microliters of the second respective mixtures thereof were added, respectively; and 15, 5 and 5 microliters of the RNA samples were added, respectively. The total volumes of the resulting reaction mixtures were 50, 20 and 20 microliters, respectively.
The contents of Kit-A and the content of Kit-B are indicated in the Table 1 and Table 2 below, respectively.
Table 1. Content of Kit-A
11
Table 2. Content of Kit-B
Content for the Kit-C for 100 tests is indicated as follows: 1000 microliters of RT-PCR 2X MasterMix, 500 microliters of S-gene/GAPDH Mix and 20 microliters of S-gene/GAPDH Positive Control.
5 to 8 microliters (here: 4 microliters) of the first mixture and 3 to 5 microliters (here: 4 microliters) of the second mixture of the kit within the scope of the present invention were used, these are combined with 4 microliters of the RNA sample; as a result, a relatively small total volume of the reaction mixture, such as 12 microliters, was worked out.
A volumetric comparison of the exemplary uses of the first and second mixtures of each kit, with the kit within the scope of the present invention, is presented in the Table 3 below.
Table 3. Respective volumetric quantities of the first and second mixtures of Kit-A, Kit-B, and Kit-C, the first and second mixtures in a preferred embodiment of the kit according to the present invention and the corresponding RNA samples (i.e., the liquid sample mixture corresponding to the mixture of the VTM with the biological sample therein), comparative to the kit according to the present invention.
Example 3- RT-PCR Protocol
The protocol applied with the kit according to the present invention is directed to conversion of the viral RNA into complementary DNA (cDNA) with the reverse transcriptase enzyme, followed by amplification of the cDNA in a polymerase chain reaction (PCR) in three steps. The events which occurred during the protocol can be summarized as follows:
1. denaturation of cDNA,
2. binding of the primers and probe to the respective denatured cDNA strands,
3. extension or synthesis of the RNA replicates by the DNA polymerase enzyme.
12
The analyzer is set according to the temperature program shown in the Table 4. The reaction mixtures in the plates or strips that are placed in the analyzer are: first, kept at 50°C to 60°C (here, at 50°C) for at least 5 minutes (here, 5 minutes), then, kept at 95°C for 10 to 20 seconds (here, 10 seconds), then, exposed to the following temperature cycle for 35 times: o 95°C for 1 to 5 seconds (here, 1 seconds) and o 60°C to 65°C (here, 60°C) for 1 to 5 seconds (here, 5 seconds).
Upon completion of said 35 cycles, the total application time of the temperature program (result time) (including the dwell times for device-based cycle transition temperature changes, in accordance with the Biorad CFX-96 Touch analyzer operated by Bio-Rad CFX Maestro Software) was 33 minutes in gross; the net total time (reaction time) of the temperature program was determined as 8.67 minutes. The processes in which the reaction mixture temperature is kept at 95°C in the cycles are directed to cDNA denaturation, thereby obtaining denatured cDNA strands (thus, RNA replicates). The processes in which the temperature is kept at 60°C are directed to the primer and probe binding to the denatured cDNA strands, and to the amplification by DNA polymerase. The cDNA amplified in these temperature cycles, creates a higher number of RNA replicates with each cycle. The probe generates a signal with a fluorescent radiation during the cycles, allowing the amount of the RNA replicates to be measured. The emission amount of the fluorescent radiation provides real-time information about the presence and amount of the target genomic material (i.e., SARS-CoV-2) obtained.
Table 4. RT-qPCR temperature program (protocol) in the method according to the present invention
The total application times of the temperature program to the respective reaction mixtures for Kit-A, Kit-B and Kit-C are 80, 65 and 60 minutes, respectively; and the temperature programs for each of these are respectively presented in Table 5, Table 6 and Table 7 below.
Table 5. Temperature program (protocol) applied with the Kit-A
13
Table 6. Temperature program (protocol) applied with the Kit-B
Table 7. Temperature program (protocol) applied with the Kit-C
Example 4 - Results
The sensitivity values in percents were calculated from the formula [GP/(GP+YN)]x100, and the specificity values in percents were calculated from the formula [GN/(GN+YP)]x100. In the formulas, the abbreviations GP, YN, GN and YP correspond to the true positive, false negative, true negative and false positive, respectively.
Example 4.1 - Results obtained with the kit according to the present invention
The kit according to the present invention detected all of 384 positives as positive, while it detected all of 400 negatives as negative. The calculated sensitivity and specificity values are presented in the Table 5 below. Table 5. Sensitivity and specificity values obtained with the kit according to the present invention
Value (%) Confidence Interval Sensitivity 100% 99.04% - 100%
Specificity 100% 99.08% - 100%
Example 5- _ Results obtained with the other kits
In a study of 100 positive and 100 negative samples:
- The sensitivity obtained with Kit-A was determined as 95.88% with a confidence interval of 89.78% to 98.87%; the specificity was determined as 93.20% with a confidence interval of 86.5% to 97.72%. The
14 number of true positives was 93, the number of false positives was 7, the number of true negatives was 96, and the number of false negatives was 4.
- The sensitivity obtained with Kit- B was determined as 96.94% with a confidence interval of 91 .31 % to 99.36%; the specificity was determined as 95.10% with a confidence interval of 88.93% to 98.39%. The number of true positives was 95, the number of false positives was 5, the number of true negatives was 97, and the number of false negatives was 3.
- The sensitivity obtained with Kit- C was determined as 94.95% with a confidence interval of 88.61 % to 98.34%; the specificity was determined as 94.06% with a confidence interval of 87.52% to 97.79%. The number of true positives was 94, the number of false positives was 6, the number of true negatives was 97, and the number of false negatives was 3.
The extraction step which is normally applied to the sample as a preliminary step in the detection of SARS- CoV-2 by real-time PCR during the pandemic period has some difficulties such as the supply of extraction kits, the additional cost that is brought by said kits, long extraction times, lack of a uniform extraction procedure, experience requirementin relation with the respective procedure, and chemical damage to the genomic material caused by lysis-based chemical methods. Due to such difficulties in the extraction process, a sample can be subjected to RT-PCR procedure as a simple, easy, cost-effective and quick solution, without any pretreatment for extraction. However, in this case, inhibition may be observed in some amplification systems and/or analytical sensitivity of the test may change, since the relevant pretreatment step is omitted from the respective method. Thanks to the kit according to the present invention, a rapid, highly accurate and cost-effective SARS-CoV-2 diagnosis is rendered possible even when an RNA extraction procedure is not performed.
References
1. C. Wu, Y. Liu, Y. Yang, P. Zhang, W. Zhong, Y. Wang, Q. Wang, Y. Xu, M. Li, X. Li, M. Zheng, L. Chen, H. Li. Analysis of therapeutictargets for SARS-CoV-2 and discovery of potential drugs bycomputational methods. Acta Pharmaceutica Sinica B, 10 (2020), pp. 766-788.
2. Kumar S, Nyodu R, Maurya VK, Saxena SK. Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Coronavirus Disease 2019 (COVID-19). 2020;23-31. Published 2020 Apr 30.
3. Cheng VC, Lau SK, Woo PC, Yuen KY. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev. 2007;20(4):660-694.
4. Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D. K., Bleicker, T., BrUnink, S., Schneider, J., Schmidt, M. L., Mulders, D. G., Haagmans, B. L., van der Veer, B., van den Brink, S., Wijsman, L., Goderski, G., Romette, J. L., Ellis, J., Zambon, M., Peiris, M., Drosten, C. (2020). Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
5. Jung, Y., Park, G. S., Moon, J. H., Ku, K., Beak, S. H., Lee, C. S., Kim, S., Park, E. C., Park, D., Lee, J. H., Byeon, C. W„ Lee, J. J., Maeng, J. S., Kim, S. J., Kim, S. I., Kim, B. T„ Lee, M. J., & Kim, H. G. (2020). Comparative Analysis of Primer-Probe Sets for RT-qPCR of COVID-19 Causative Virus (SARS-CoV-2). ACS infectious diseases, 6(9), 2513-2523.
15
6. CDC (2020) 2019-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Panel: Primers and Probes, https://www.who.int/docs/default-source/coronaviruse/uscdcrt-pcr-panel-primer-probes.pdf (accessed on 12.05.2021)
7. Mollaei, H. R., Afshar, A. A., Kalantar-Neyestanaki, D., Fazlalipour, M., & Aflatoonian, B. (2020). Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR. Iranian journal of microbiology, 12(3), 185-193.
8. Coronavirus Variants and Mutations, By Jonathan Corum and Carl Zimmer, https://www.nytimes.com/interactive/2021/health/coronavirus-variant-tracker.html (accessed on 13.05.2021)
16
—SEQUENCE LISTING—
<110> TURKIYE SAGLIK ENSTITULERI BASKANLIGI
<120> A METHOD AND KIT FOR QUANTITATIVE DETECTION OF SARS-CoV-2 BY REAL-TIME PCR <160> 6
<210> SEQ ID NO: 1 <211 > 22 <212> DNA <213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide <400> 1 gggagccttg aatacaccaa aa
<210> SEQ ID NO: 2 <211 > 21 <212> DNA <213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide <400> 2 tgtagcacga ttgcagcatt g
<210> SEQ ID NO: 3 <211 > 24 <212> DNA <213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide <400> 3 atcacattgg cacccgcaat cctg
<210> SEQ ID NO: 4 <211 > 19 <212> DNA <213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide <400> 4 agatttggac ctgcgagcg
<210> SEQ ID NO: 5 <211 > 20 <212> DNA <213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide <400> 5 gagcggctgt ctccacaagt
<210> SEQ ID NO: 6 <211 > 23 <212> DNA <213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide <400> 6 ttctgacctg aaggctctgc gcg
Claims
1. A SARS-CoV-2 test kit for real-time RT-PCR, wherein said kit includes a first mixture, comprising a primer set that includes a first forward primer (SEQ ID NO: 1 ) with 5’-GGGAGCCTTGAATACACCAAAA- 3’ structure corresponding to positions 28.320 to 28.376 remaining in N gene region of SARS-CoV-2 RNA and a first reverse primer (SEQ ID NO: 2) with 5’-TGTAGCACGATTGCAGCATTG-3’ structure.
2. The test kit according to claim 1 , wherein said first mixture comprises a first fluorogenic probe (SEQ ID NO: 3) with 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure and a fluorescent dye at 5' terminus and a quencher dye at 3' terminus.
3. The test kit according to any one of claims 1 or 2, wherein said first mixture comprises a second forward primer (SEQ ID NO: 4) with 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5) with 5’-GAGCGGCTGTCTCCACAAGT-3’ structure and a second fluorogenic probe (SEQ ID NO: 6) with 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure.
4. The test kit according to any one of claims 1 to 3, wherein said kit includes a second mixture that comprises a reagents mixture comprising one or more buffers, one or more cofactors, more than one nucleotides, and one or more enzymes.
5. The test kit according to claim 4, wherein said second mixture is a reagents mixture comprising one or more buffers, Mg2+ as a cofactor, deoxyribonucleotide triphosphate as one of said nucleotides, one or more ribonuclease inhibitors, one or more reverse transcriptases, and one or more Taq polymerases.
6. A method for the quantitative detection of the presence of SARS-CoV-2 in a biological sample, wherein said method comprises the following steps:
- detection of a presence of a SARS-CoV-2 RNA in a reaction mixture obtained from a biological sample by quantitative real-time PCR with the following steps; a) amplification of a cDNA corresponding to the SARS-CoV-2 RNA with a first forward primer (SEQ
ID NO: 1 ) with 5’-GGGAGCCTTGAATACACCAAAA-3’ structure and a first reverse primer (SEQ ID NO: 2) with 5’-TGTAGCACGATTGCAGCATTG-3’ structure, b) detecting, simultaneously with step (a), an amplification amount of cDNA by using a first fluorogenic probe (SEQ ID NO: 3) with 5’-ATCACATTGGCACCCGCAATCCTG-3’ structure and a fluorescent dye at the 5' terminus and a quencher dye at the 3' terminus.
7. The method according to claim 6, wherein said method comprises a comparison of the amplification amount of cDNA with at least one control sample.
8. The method according to any one of claims 6 or 7, wherein said method includes subjecting the reaction mixture to a mixing process before the step (a) without implementation of an RNA extraction procedure.
9. The method according to claim 8, wherein said mixing is performed by using a vortex device.
10. The method according to any one of claims 6 to 9, wherein said method includes obtaining said reaction mixture by mixing the following with each other:
18 a first mixture that includes a first forward primer (SEQ ID NO: 1) with 5’- GGGAGCCTTGAATACACCAAAA-3’ structure corresponding to positions 28.320 to 28.376 remaining in an N gene region of SARS-CoV-2 RNA and a first reverse primer (SEQ ID NO: 2) with 5’- TGTAGCACGATTGCAGCATTG-3’ structure, a second mixture that includes a reagents mixture comprising one or more buffers, one or more cofactors, more than one nucleotides, and one or more enzymes, and said biological sample
11. The method according to claim 10, wherein said method includes obtaining said reaction mixture by mixing the following with each other: a first mixture that includes a primer set comprising a first forward primer (SEQ ID NO: 1 ) with 5’- GGGAGCCTTGAATACACCAAAA-3’ structure and a first reverse primer (SEQ ID NO: 2) with 5’- TGTAGCACGATTGCAGCATTG-3’ structure, a second mixture that includes a reagents mixture comprising one or more buffers, Mg2+ as a cofactor, more than one nucleotides, one or more ribonuclease inhibitors, one or more reverse transcriptases, and one or more Taq polymerases, and said biological sample.
12. The method according to any one of claims 10 or 11 , wherein said method includes addition of a second forward primer (SEQ ID NO: 4) with 5’-AGATTTGGACCTGCGAGCG-3’ structure, a second reverse primer (SEQ ID NO: 5) with 5’-GAGCGGCTGTCTCCACAAGT-3’ structure, and a second fluorogenic probe (SEQ ID NO: 6) with 5’-TTCTGACCTGAAGGCTCTGCGCG-3’ structure into the reaction mixture.
13. The method according to any one of claims 6 to 13, wherein said method includes subjecting the reaction mixture to the following temperature protocol in the real-time RT-PCR test: iv. keeping the reaction mixture at 50°C to 60°C for at least 5 minutes, v. keeping the reaction mixture at 95°C for 10 to 20 seconds after step i, vi. subjecting the reaction mixture to the following temperature cycles 35 times after step ii: 95°C for 1 to 5 seconds and then 60°C to 65°C for 1 to 5 seconds.
14. The method according to claim 13, wherein said method comprises subjecting the reaction mixture to the following temperature protocol in the real-time RT-PCR test: i. keeping the reaction mixture at 50°C for 5 minutes, ii. keeping the reaction mixture at 95°C for 10 seconds after step i, iii. subjecting the reaction mixture to the following temperature cycles 35 times after step ii: 95°C for 1 seconds and then 60°C for 5 seconds.
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Non-Patent Citations (2)
| Title |
|---|
| WON, J. ET AL.: "Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19", EXP NEUROBIOL, vol. 29, 30 April 2020 (2020-04-30), pages 107 - 119, XP055806478, DOI: 10.5607/en20009 * |
| ZHANG XUZHAO, LI MIN, ZHANG BIN, CHEN TAO, LV DONG, XIA PENGFEI, SUN ZHUANYI, SHENTU XIAOYAN, CHEN HAIYAN, LI LIBIN, QIAN WENBIN: "The N gene of SARS-CoV-2 was the main positive component in repositive samples from a cohort of COVID-19 patients in Wuhan, China", CLINICA CHIMICA ACTA, ELSEVIER BV, AMSTERDAM, NL, vol. 511, 1 December 2020 (2020-12-01), AMSTERDAM, NL , pages 291 - 297, XP093007030, ISSN: 0009-8981, DOI: 10.1016/j.cca.2020.10.019 * |
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