WO2021245708A1 - Procédé rapide et sensible de détection de sras-cov-2 - Google Patents

Procédé rapide et sensible de détection de sras-cov-2 Download PDF

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WO2021245708A1
WO2021245708A1 PCT/IN2021/050549 IN2021050549W WO2021245708A1 WO 2021245708 A1 WO2021245708 A1 WO 2021245708A1 IN 2021050549 W IN2021050549 W IN 2021050549W WO 2021245708 A1 WO2021245708 A1 WO 2021245708A1
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primers
gene
seq
cov
sars
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WO2021245708A4 (fr
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Shyam Sundar NANDI
Upendra Pradeep LAMBE
Sonali Ankush Sawant
Trupti Shashikant GOHIL
Jagadish Mohan DESHPANDE
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Indian Council Of Medical Research
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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
    • C12Q1/701—Specific hybridization probes

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  • the present invention generally relates to the field of molecular biology. Particularly, the present invention relates to a method of detecting SARS-CoV-2. More specifically, the present invention provides a rapid and sensitive method for detecting SARS-CoV-2 in biological samples. The present invention is specific and based on RT-LAMP reaction.
  • Severe acute respiratory syndrome -related coronavirus is a species of coronavirus that infects humans, bats and certain other mammals. It is an enveloped positive-sense single-stranded RNA virus and a member of the genus Betacoronavirus and subgenus Sarbecovirus.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS severe respiratory disease 2019
  • respiratory problems such as coughing and difficulty of breathing.
  • symptoms such as headache, shaking chills, loss of appetite, generalized malaise, diarrhea, or clouding of consciousness are observed .
  • these symptoms are almost the same as those of other respiratory diseases, such as influenza.
  • COVID-19 is an infectious disease caused by SARS-CoV-2 that began in Wuhan, China in December 2019, and has caused serious infection across the globe. According to the World Health Organization (WHO), the mortality for the patients afflicted with SARS is deduced to be 3.4% on average (WHO situation report 129).
  • the SARS-CoV-2 which is a pathogenic virus of SARS, is a single- stranded RNA virus.
  • rRT-PCR reverse transcription real time RT-PCR
  • kits are approved by government agencies for rRT-PCR.
  • the available molecular detection assay is a robust and reliable method for detection of many pathogens. However, even though the assay is quite specific and sensitive, but the time required for detection and the sophisticated instrumentation required along with the skilled professionals limits its usage in pandemic situation.
  • the present invention address the above problems and provides a method which is rapid as well as sensitive for the detection of SARS-CoV-2.
  • a rapid and sensitive method for detecting presence of SARS-CoV-2 comprising of: i. providing a biological sample and extracting RNA; ii. adding the RNA sample of step (i) to a set of loop-mediated isothermal amplification (LAMP) primers specific for genes selected from envelope protein (E), membrane glycoprotein (M), spike glycoprotein (S), nucleocapsid protein (N) and RNA dependent RNA polymerase (RdRp); wherein the set of primers are selected from group consisting of SEQ ID NOs: 1-50; iii. incubating the sample of step (ii) with a colorimetric LAMP reagent in a reaction mixture at a temperature of 65°C for 30 minutes; and iv. obtaining a visual change in the reaction mixture.
  • LAMP loop-mediated isothermal amplification
  • the set of primers for envelope protein (E) gene is selected from the group consisting of SEQ ID NO. 1-9.
  • the set of primers for membrane glycoprotein (M) gene is selected from the group consisting of SEQ ID NO. 10-19.
  • the set of primers for spike glycoprotein (S) gene is selected from the group consisting of SEQ ID NOs: 20-28.
  • nucleocapsid protein (N) gene is selected from the group consisting of SEQ ID NO. 29-40.
  • RNA dependent RNA polymerase (RdRp) gene is selected from the group consisting of SEQ ID NO. 41-50.
  • genes are selected from envelope protein (E) gene and nucleocapsid protein (N) gene.
  • primers specific for envelope protein (E) gene and nucleocapsid protein (N) gene is selected from SEQ ID NOs: 5-9 and SEQ ID NO. 35-40.
  • the biological sample is a throat swab, nasal swab and sewage sample.
  • primers having a nucleic acid sequences selected from SEQ ID NO. 1-50 useful for detecting the presence of SARS-CoV-2.
  • primers selected from SEQ ID NO. 5-9 and SEQ ID NO. 35-40.
  • Figure 1 illustrates workflow of RT-LAMP based method for detection of SARS- CoV-2.
  • Figure 2 illustrates the working of all the ten LAMP primer sets. The results can be interpreted as; pink color (no amplification occurred, no proton released, no pH- dependent color change within 30 minutes at 65°C) in reaction tube is negative amplification; and color change from pink to yellow indicates positive amplification (amplification occurred, proton released, pH-dependent color change from pink to yellow, within 30 minutes at 65°C.)
  • Figure 3a illustrates the results of 8 clinical samples along with one Negative Test Control (NTC) and one Positive Test Control (PTC).
  • Five RT LAMP primers namely, E gene setl (El), E gene set2 (E2), M gene set4 (M4), M gene set4b (M4b) and S gene set2 (S2) were used.
  • the samples having Ct value ranging from 20 - 35 Ct value were used.
  • Figure 3b illustrates the results of 8 clinical samples along with one Negative Test Control (NTC) and one Positive Test Control (PTC).
  • NTC Negative Test Control
  • PTC Positive Test Control
  • the samples having Ct value ranging from 20 - 35 Ct value were used.
  • Figure 4 illustrates the results obtained by E2 and N5 primers by RT-LAMP based method for detection of SARS-CoV-2. The results can be interpreted as; pink color (no color change) in reaction tube is negative amplification and yellow color change is positive amplification.
  • Both E and N gene reaction tubes are pink- Negative for both E and N gene;
  • E gene reaction tube is pink and N gene reaction tube is yellow- Negative for E gene and positive for N gene;
  • E gene reaction tube is yellow and N gene reaction tube is pink- Positive for E gene and negative for N gene and
  • Both E and N gene reaction tubes are yellow- Positive for both E and N gene.
  • Figure 5 illustrates the results of limit of detection based on the E gene clone of SARS- CoV-2.
  • Ten-fold serial dilution was performed upto 10 15 .
  • the reaction tube of serial silution 10 1 to 10 11 showed yellow coloration indicating positive result. While the reaction tube of serial dilution 10 12 to 10 15 showed pink colouration indicating negative result.
  • the initial copy number for 10 1 dilution tube was 2.83xl0 u and it was detected upto 10 1 copy number. 4pl of the RNA sample was used in the reaction tube.
  • the limit of detection for E gene clone was found to be upto 40 copy number per reaction.
  • Figure 6 illustrates the results of limit of detection based on the N gene clone of SARS- CoV-2.
  • Ten-fold serial dilution was performed upto 10 15 .
  • the reaction tube of serial silution 10 1 to 10 11 showed yellow coloration indicating positive result. While the reaction tube of serial dilution 10 12 to 10 15 showed pink colouration indicating negative result.
  • the initial copy number for 10 1 dilution tube was 3xl0 u and it was detected upto 10 1 copy number. 4m1 of the RNA sample in reaction tube, thus the limit of detection for N gene clone was found to be upto 40 copy number per reaction.
  • the present invention relates to a rapid, sensitive and specific method that targets SARS-CoV-2.
  • the present invention provides a rapid and sensitive method for detecting SARS-CoV-2 in biological samples.
  • the method is based on the use of specific primer combination for the detection and the reaction is incubated at a single temperature (isothermal reaction at 65°C) for 30 minutes. Further, an important aspect of the method is colorimetric detection, thus making it easy and convenient to have rapid visual results with high accuracy.
  • the diagnostic sensitivity and specificity of RT-LAMP is 98.46% and 100%, respectively, considering rRT-PCR as a gold standard.
  • the method of the present invention is based on Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) reaction by designing two sets of specific RT-LAMP primers targeting regions (S gene, M gene, E gene, N gene and RdRp gene) of SARS-CoV-2 genome. Further, the principle of this method is based on pH sensitive dyes to exploit the change in the pH resulting from proton accumulation while incorporation of dNTPs. The change in the color of reaction can be detected by naked eyes and leads to rapid and convenient detection.
  • reaction setup includes colorimetric RT-LAMP reagent, primer mix, enhancer solution (1M guanidine hydrochloride) and extracted RNA followed by incubation at 65°C for 30 minutes. The interpretation of results is done by observing color change. Yellow color indicates positive result, while pink color indicates negative results.
  • a rapid and sensitive method for detecting presence of SARS-CoV-2 comprising of: i. providing a biological sample and extracting RNA; ii. adding the RNA sample of step (i) to a set of loop-mediated isothermal amplification (LAMP) primers specific for genes selected from envelope protein (E), membrane glycoprotein (M), spike glycoprotein (S), nucleocapsid protein (N) and RNA dependent RNA polymerase (RdRp); wherein the set of primers are selected from group consisting of SEQ ID NOs: 1-50; iii. incubating the sample of step (ii) with a colorimetric LAMP reagent in a reaction mixture at a temperature of 65°C for 30 minutes; and iv. obtaining a visual change in the reaction mixture.
  • LAMP loop-mediated isothermal amplification
  • a method for detecting presence of SARS-CoV-2 wherein the set of primers for envelope protein (E) gene is selected from the group consisting of SEQ ID NO. 1-9.
  • a method for detecting presence of SARS-CoV-2 wherein the set of primers for membrane glycoprotein (M) gene is selected from the group consisting of SEQ ID NO. 10-19.
  • a method for detecting presence of SARS-CoV-2 wherein the set of primers for spike glycoprotein (S) gene is selected from the group consisting of SEQ ID NOs: 20-28.
  • a method for detecting presence of SARS-CoV-2 wherein the set of primers for nucleocapsid protein (N) gene is selected from the group consisting of SEQ ID NO. 29-40.
  • a method for detecting presence of SARS-CoV-2 wherein the set of primers for RNA dependent RNA polymerase (RdRp) gene is selected from the group consisting of SEQ ID NO. 41-50.
  • the genes are selected from envelope protein (E) gene and nucleocapsid protein (N) gene.
  • a method for detecting presence of SARS-CoV-2 wherein the primers specific for envelope protein (E) gene and nucleocapsid protein (N) gene is selected from SEQ ID NOs: 5-9 and SEQ ID NO. 35-40.
  • a method for detecting presence of SARS-CoV-2 wherein the biological sample is a throat swab, nasal swab and a sewage sample.
  • primers having a nucleic acid sequences selected from SEQ ID NO. 1-50 useful for detecting the presence of SARS-CoV-2.
  • the primers are selected from SEQ ID NO. 5-9 and SEQ ID NO. 35-40.
  • the method of the present invention has various advantages such as, the method does not require sophisticated instruments for the performance and results interpretation.
  • the time required for performing the method is very less i.e. around 30 minutes.
  • the method can be performed using dry bath/water bath (65 ⁇ 1°C) and the results can be interpreted visually.
  • the present method does not require highly skilled or technical person and is cost effective, convenient and rapid.
  • a kit can be developed based on the method of the present invention, which would be suitable to be performed as a point of care diagnostic assay at the health care centers, surveillance laboratories, airports, frontier ports and other areas and has important significance for preventing and controlling the spread of Coronavirus disease- 19 (COVID-19).
  • the diagnostic sensitivity and specificity of the present method was 98.46% and 100%, respectively, considering rRT-PCR as a gold standard.
  • the SARS-CoV-2 (Accession number NC_045512.2) specific conserved regions were identified for primer designing.
  • the present method is based on RT-LAMP reaction and uses multiple forward and reverse (backward) primers and one or two loop primers.
  • the method involves using RTx reverse transcriptase that gets activated above 45°C and modified Bst 2.0 DNA polymerase having strand displacement activity. Further, RT-LAMP reactions can therefore, be carried out at a single incubation temperature (isothermal).
  • the primers B2 and B3 initiate cDNA synthesis
  • FIP and BIP are specialized primers consisting of two parts, F2/Flc and B2/Blc, respectively.
  • F2 and B2 bind with the template strand to initiate amplification process, while Flc and Blc sequences serve as overhangs which help loop formation as RT-LAMP reaction continues.
  • the short distance between the F2 and Flc (and B2/ Blc) helps formation of a loop structure within the amplicon.
  • the loop primers increase the number of initiation points for DNA synthesis by binding complementarily to the single stranded loops and increase the pace of amplification.
  • the four key factors in the LAMP primer designing were the melting temperature (Tm), stability at the 3’ and 5’ end of each primer (Delta G), GC content and ability to form secondary structures.
  • Tm was calculated by using the Nearest-Neighbour method. This method is presently considered to be the method that predicts the Tm value closest to the actual value.
  • the Tm for each region was designed to be about 65°C (64 - 66°C) for Flc and B lc, about 60°C (59 - 61°C) for F2, B2, F3, and B3, and about 65°C (64 - 66°C) for the loop primers.
  • the 3’ end of the primers acts as the initiating point of the DNA polymerization and therefore, must be very stable and complementary with the target sequence. The following criteria was taken into consideration, while selecting the primer sets.
  • the 3’ ends of F2/B2, F3/B3 was designed, so that the free energy is -4 kcal/ mol or less.
  • LF/LB and the 5’ end of Flc/B lc was designed so that the free energy is -4 kcal/ mol or less.
  • Primers were designed so that their GC content is between about 40% to 65%. But, primers with GC content between 50% and 60% were selected as they are considered to give relatively better results.
  • primers Another important property of primers is the ability to form secondary structures or primer dimers.
  • the primers were designed so that they do not form secondary structures. To avoid this condition, it is important to make sure that the 3’ end of the primer is non complementary.
  • Example 2 Designing of primers for the genes encoding Spike glycoprotein (S), Membrane glycoprotein (M), Envelope protein (E), Nucleo-capsid protein (N) and RNA dependent RNA polymerase (RdRp)
  • the primers were synthesized by Sigma Aldrich and were received on May 22 nd 2020. All the primer sets were dissolved to make a solution of IOOmM primer concentration each. Further these primers were mixed so as to get a concentration of 16 mM for FIP and BIP, 2 pM for F3 and B3, and 4 pM for LF and LB in at 10X concentration.
  • the IX primer mixture consists of 1.6 pM of FIP and BIP, 0.2 pM of F3 and B3, and 0.4 pM of LF and LB which is used for RT-LAMP reaction (Table 2). Table 2: Concentration of each primer to be incorporated in the primer mixture.
  • the Enhancer consists of 1M solution of Guanidine hydrochloride (Sigma-Aldrich Cat. No: G3272-25G) molecular biology grade and pH of 8 was adjusted by using 1M KOH (Sigma-Aldrich Cat no:221473-25G).
  • Example 4 Isolation of Biological Samples Nasal/ throat swabs of suspected COVID-19 patients collected in the virus transport medium (VTM) by various hospitals were received for laboratory diagnosis under COVID-19 surveillance. Samples were tested by real time RT-PCR as per the ICMR standard protocol and results reported to the sender hospital, state government and the ICMR database. Further, the samples were tested retrospectively by the method based on the RT-LAMP. Patient’s demographic information and clinical details were not provided to the laboratory for retrospective testing. Bio-safety laboratory practices were strictly followed for sample processing and testing.
  • VTM virus transport medium
  • AVE Elution buffer
  • a commercially available kit was used for RT-LAMP based method which comprises WarmS tart Colorimetric LAMP 2X Master Mix with UDG (NEB Cat No. M1804L).
  • the work flow of the method for detection of SARS-CoV-2 is described in the schematic diagram ( Figure 1).
  • the 10 X concentration of primer mix for each of the 10 set of primers was used.
  • the reaction was setup by addition of all the components in a 20 pi reaction mixture (Table 3).
  • Table 3 Composition of 20 ul RT-LAMP reaction mix.
  • a reagent mix was prepared freshly for the number of samples to be included in a particular test run and 16 pi of the mixture was distributed in 200ul PCR tubes. 4 m ⁇ of test RNA sample or controls was then added. The 20 m ⁇ reaction should have pink color.
  • the reaction mixture was mixed followed by a quick spin and incubated at 65 °C for different time intervals upto 75 minutes. The results were observed with naked eye by looking at the color change. The color change was observed after every 5 minutes from 30 minutes of incubation in the thermal cycler.
  • M gene two primer sets were used M4 and M4b, but set M4 worked giving a better result.
  • N gene two primer sets were used N2 and N5. Both sets worked properly but set N5 is more stable.
  • S gene two primer sets were used S2 and S4. Both the sets worked. But S2 worked giving better results.
  • E gene two primer sets were used El and E2. Both sets were worked properly but set E2 is more stable.
  • RdRp gene two primer sets were used B4 and C4. Both the primer set worked. ( Figure 2).
  • Example 5.1 Temperature optimization and sensitivity of LAMP primers
  • M4 set of primer were used to select the broad range of temperatures. This was followed by selection of narrow range of temperatures for all the primer sets belonging to M gene, N gene, E gene, S gene and RdRp genes.
  • the target RNA was serially 10 fold diluted ranging from original RNA sample to the 10 6 dilution. The experiment was performed using all the primer set. The E2 and N5 primers were found to be more sensitive RT LAMP primers.
  • Example 5.2 Pilot testing of 25 samples
  • Example 5.3 Parallel testing of 66 samples in comparison to rRT-PCR Labgun kit
  • Example 6 Comparing sensitivity and specificity of RT-LAMP based method with RT-PCR method
  • the colorimetric RT-LAMP based method was used to test 253 clinical samples and the results were comparable with real-time RT-PCR.
  • the RT-LAMP based method was based in the detection of Envelop (E) and Nucleocapsid (N) genes. The tubes were removed from the incubator and observed with the naked eye. The positive reaction was indicated by yellow colored reaction mix. On the other hand, the color of negative reaction remained pink (Ligure 4).
  • the color change is not significant, for example if the color of reaction is orange, incubate the reaction at 65 ° C for more 10 minutes and re-examined for the complete color change. The color of reaction gets intensified by cooling the reaction to cool down at room temperature. The results indicate that the diagnostic sensitivity and specificity of the RT-LAMP based method assay was 98.46% and 100%, respectively, as compared to the rRT-PCR.
  • Table 4 represents comparative evaluation of results of RT-LAMP based method and real time RT-PCR.
  • the detection based on E gene with present method reported 130 positives, 120 negatives and 3 PCR negatives as positives (false positive). Thus, it was found that the detection based on E gene showed 100% sensitivity and 97.56% specificity with the reference standard. Further, the detection based on N gene using the present method reported 128 positives and 121 negatives (98.46% sensitivity and 98.37% specificity). Considering that a positive reaction in either of the two genes (E or N) reports a SARS-CoV-2 positivity, it resulted in 100% sensitivity with a slight loss specificity (96.75%).
  • the E and N gene of SARS-CoV-2 were cloned in TA cloning vector pTZ57R/T (Thermo Fisher Scientific Cat No. K1213) in-house.
  • the cloning primers were designed with 5’ T7 Promotor sequence overhang.
  • In-vitro transcription of the cloned fragment was done using T7 RNA polymerase (Thermo Fisher Cat No. K0441).
  • RNA transcript were purified using GeneJet RNA Clean up and Concentration Micro kit (Cat No. K0842). The concentration of RNA in PTC of E and N gene was 760 ng/pl each.
  • RNA transcripts Ten-fold serial dilutions of the RNA transcripts were used for calculating concentration and copy numbers by QubitTM RNA HS Assay kit (Cat. No. Q32852). The limit of detection for E gene and N gene was found to be 40 copy number ( Figure 5; Table 5 and Figure 6; Table 6).

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Abstract

La présente invention concerne un procédé rapide et sensible de détection du SARS-CoV-2. L'invention concerne plus particulièrement un procédé comprenant les étapes suivantes : (I) fournir un échantillon biologique et extraire l'ARN ; (ii) ajouter un échantillon d'ARN de l'étape (i) à un ensemble d'amorces à amplification isotherme à médiation par boucle (LAMP) spécifiques des gènes choisis parmi la protéine d'enveloppe (E), la glycoprotéine membranaire (M), la glycoprotéine spike (S), la protéine de nucléocapside (N) et l'ARN polymérase ARN-dépendante (RdRp, de l'anglais « RNA dependent RNA polymerase ») ; l'ensemble d'amorces étant choisi dans le groupe constitué par SEQ ID No : 1 à 50 ; (iii) incuber l'échantillon de l'étape (ii) avec un réactif LAMP colorimétrique dans un mélange réactionnel à une température de 65°C pendant 30 minutes ; et (iv) obtenir un changement visuel dans le mélange réactionnel. Le présent procédé est spécifique et les résultats peuvent être observés à l'œil nu.
PCT/IN2021/050549 2020-06-05 2021-06-05 Procédé rapide et sensible de détection de sras-cov-2 WO2021245708A1 (fr)

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