WO2005059177A1 - A sensitive and specific test to detect sars coronavirus - Google Patents
A sensitive and specific test to detect sars coronavirus Download PDFInfo
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- WO2005059177A1 WO2005059177A1 PCT/SG2004/000416 SG2004000416W WO2005059177A1 WO 2005059177 A1 WO2005059177 A1 WO 2005059177A1 SG 2004000416 W SG2004000416 W SG 2004000416W WO 2005059177 A1 WO2005059177 A1 WO 2005059177A1
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- 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
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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
Definitions
- Sequence Listing The present application includes an appended Sequence Listing of 20 pages presenting 12 sequences.
- the instant invention provides a qualitative nucleic acid amplification assay for the detection of SARS coronavirus in patient samples.
- the assay uses primer pairs that have been developed that provide excellent sensitivity and specificity for detection of SARS coronavirus.
- SARS coronavirus SARS coronavirus
- SARS RT-PCR based diagnostic tests often suffer the drawback of being complex and difficult to administer.
- the typical SARS diagnostic test uses nested (two step) polymerase chain reaction (PCR) to accomplish a certain level of specificity and sensitivity. See, e.g., "SARS-CoV Specific RT-PCR Primers", by William J. Bellini, Ph.D.
- Figure 1 shows the portions of the SARS coronavirus genome amplified by the IMCB primer sets.
- Figure 2 shows the portion of the SARS coronavirus genome amplified by the IMCB-3 primer set and aligns the IMCB-3 primers and the IMCB-3 probe along the sequence of the SARS coronavirus genome.
- the upper strand sequence is shown as nucleotides 4609-4765 of SEQ ID NO: 1.
- the lower strand is shown as SEQ ID NO: 12.
- Figures 3A-3C show gels demonstrating the efficacy of the primers of the invention.
- Figure 3A shows the detection of SARS coronavirus using the IMCB-2 primer set where the virus copy number per sample loaded varies from 26.1 copies to 0.07 copies.
- Lane 1 is a marker
- lanes 2 & 3 contain 26.1 copies of the virus per 5 ⁇ l
- lanes 4 & 5 contain 12.6 copies of the virus per 5 ⁇ l
- lanes 6 & 7 contain 1.96 copies of the virus per 5 ⁇ l
- lanes 8 & 9 contain 2.0 copies of the virus per 5 ⁇ l
- lanes 10 & 11 contain 0.07 copies of the virus per 5 ⁇ l
- lane 12 contains a negative control of an unrelated virus
- lane 13 contains another marker.
- Figure 3B shows a second experiment detecting SARS coronavirus using the IMCB-2 primer set, where the virus copy number per sample loaded varies from 26.1 copies to 0.08 copies.
- Lane 1 is a marker
- lanes 2 & 3 contain 26.1 copies of the virus per 5 ⁇ .1
- lanes 4 & 5 contain 8.2 copies of the virus per 5 ⁇ l
- lanes 6 & 7 contain 2.6 copies of the virus per 5 ⁇ l
- lane 8 contains 0.8 copies of the virus per 5 ⁇ l
- lane 9 contains 0.25 copies of the virus per 5 ⁇ l
- lanes 10 & 11 contain 0.O8 copies of the virus per 5 ⁇ l
- lane 12 contains a negative control of an unrelated virus
- lane 13 contains another marker.
- Figure 3C shows the amplified product from a sample containing 5 copies of SARS coronavirus genomic RNA per run in a total of 5 ⁇ l (duplicated) .
- FIG. 1 The product is resolved by 3% agarose gel electrophoresis. A 10% of total reaction volume (5 ⁇ l) is loaded per: lane. Lane 1, amplified product; lane 2, amplified product of a duplicate reaction; lane M, 100 bp ladder.
- Figures 4A to 4C show the sensitivity achieved using the present invention to detect SARS coronavirus nucleic acid with the primer set IMCB-1. Figure 4A shows results achieved with 8.8 pfu (2200 copies) per sample (lanes 1 and 2) to 0.08 pfu (22 copies) per sample (lanes 5-6). Lanes 7 and 8 show a no virus control.
- Figure 4B shows the results of another run of the same assay using from 0.08 pfu (22 copies) per sample to 0.0008 pfu (0.2 copies) per sample. Lanes 11 and 12 show a no virus control .
- Figure 4C shows a third run using from 0.08 pfu (22 copies) per sample to 0.004 pfu (1 copy) per sample. Lanes 7 and 8 are a no virus control sample.
- M Is a molecular length marker.
- SARS coronavirus SARS coronavirus
- Coronoviruses are a family of RNA viruses with a large envelope that propagate in the cytoplasm of host cells and usually cause mild respiratory disease in man and animals.
- the SARS Coronavirus has been isolated and sequenced. A prototype sequence of 29,727 basepairs can be found at GENBANK, under Accession No. AY278741, hereby incorporated by reference and presented also as SEQ ID NO: 1. See also, Y.J.
- the SARS virus encodes 14 open reading frames (ORFs) , including the replicase la and lb proteins and four structural proteins, spike protein (S) , envelope protein (E) , membrane protein (M) and nucleocapsid protein (N) .
- ORFs open reading frames
- S spike protein
- E envelope protein
- M membrane protein
- N nucleocapsid protein
- Ruan et al . compared the genome sequence of a Singapore case of SARS coronavirus and a database of other coronavirus genomic sequences. From this they were able to find which regions of SARS coronavirus were homologous to other coronavirus and thus conserved among coronavirus strains and which sequences were unique to SARS coronavirus Singapore strain SIN2500.
- the instant invention provides a simple, sensitive and specific diagnostic test.
- This test provides a simple yet sensitive and specific nucleic acid amplification system compared with others that have so far been developed.
- the instant invention is made more sensitive and specific than the detection methods of the prior art.
- specificity and sensitivity may be enhanced by using a one step PCR method, instead of a two step PCR.
- the present invention utilizes specific primer pairs designed from the SARS coronavirus non-structural protein 1 (NSPl) a putative proteinase . These primers can be used with many techniques to detect the presence of SARS coronavirus .
- the instant invention provides a simple gel-based RT-PCR detection kit.
- kits will include one or more primers and/or probes according to the invention, for example a kit may contain primers consisting of one or more polynucleotides comprising a nucleotide sequence of SEQ ID NOs : 3, 4, 6, 7, 9, 10 and 11.
- a kit according to the invention may optionally include a positive control nucleic acid, for example a SARS coronavirus genomic nucleic acid, or at least a portion thereof comprising the NSPl region, as either RNA or DNA.
- the present invention also provides a method for detecting SARS coronavirus nucleic acid in a sample.
- the method may be generally described as comprising amplifying a nucleic acid of a sample with a reverse transcriptase and at least one primer specific for the NSPl region of a SARS coronavirus to generate a nucleic acid ampification product.
- the amplification product is then analyzed, seeking to detect an expected nucleic acid amplification product. Detection of the expected product indicates the presence of SARS coronavirus nucleic acid in the sample.
- the primers of the instant invention can also be used as a primer set for real-time PCR detection using PCR platforms such as the Roche LightCycler;TM, the Stratagene Real-time PCR system, the Applied Biosystems ABI 7000 real time PCR analyzer or any other suitable detection platform.
- primer pairs have been designed by comparing conserved regions among SARS coronavirus strains and avoiding sequence regions that were conserved among all coronaviruses generally.
- a portion of the SARS coronavirus genome that encodes the NSPl proteinase was selected by this comparison as a detection target.
- the primers of the present invention hybridize in the portion of the SARS coronavirus genome from about nucleotide number 2200 to about nucleotide 9800 in a manner as to amplify this region, or a portion thereof. This region is known as the NSPl region, which codes for a putative proteinase.
- This region was chosen as a putative region to amplify because unlike the RNA polymerase region utilized in prior art assays, there is a significant portion of it in which no mutations appear to have occurred among SARS coronaviruses and this region is very specific to all isolates of SARS coronavirus; a hypothesis which has been examined by an NCBI (National Center for Biotechnology Information) Blast search. In the region from about nucleotide 2650 to about nucleotide 7850 has been identified that appears to bear no mutations among several strains (Ruan et al . , 2003). A preferred part of the NSPl region for amplification is the part from and including nucleotide 4609 to and including nucleotide 7003.
- the primers of the invention should be at least 16 nucleotides in length, more preferably at least 18 nucleotides in length, still more preferably at least 20 nucleotides in length.
- the primers should be less than 50, preferably less than 30, more preferably less than 25 nucleotides in length, so as to preserve the specificity of the primers .
- the SARS detection method of the invention lies generally in use of a set of primers that are specific for the NSPl region of the SARS coronavirus genome for PCR amplification of this part of the genome and detection of the amplification product .
- the method of the invention can be performed, for example by amplifying nucleic acids present in the sample using a forward primer and a reverse primer selective for the region of the SARS genome from nucleotide 6652 to nucleotide 7003, or using a forward primer and a reverse primer selective for the region of the SARS genome from nucleotide 4609 to nucleotide 4765, said primers having a certain primer length in nucleotides and being separated by a separation length that is a certain number of nucleotides, to obtain an amplification product. The amplification product is then detected.
- the amplification product can be detected, for example, by determining the length of the amplification product in nucleotides, either by a chromatographic method or by a gel electrophoretic method, e.g by electrophoresis in 2 or 3% agarose .
- the presence of an amplification product having a length in nucleotides that is the sum of the forward primer length, the reverse primer length and the separation length indicates the presence of SARS coronavirus nucleic acid in the sample.
- the product can be detected using a hybridization probe, for example "using real-time fluorescent detection in the TaqmanTM system.
- the hybridization probe preferably comprises a nucleotide sequence that is the same as that of a portion of the amplification product that would be obtained using the amplification primers selected and a SARS coronavirus genomic nucleic acid as a template.
- the hybridization probe should be at least 16 nucleotides in length, more preferably at least 18 nucleotides in length, still more preferably at least 20 nucleotides in length.
- the probe should be less than 50 , preferably less than 30, more preferably less than 25 nucleotides in length, so as to preserve the specificity of the probe .
- a primer or probe according to the present invention The essential function of a primer or probe according to the present invention is to specifically hybridize to a SARS coronavirus nucleic acid, either an RNA or DNA, and not to cross-hybridize to other coronavirus nucleic acids or to nucleic acids of other viruses.
- a primer or probe according to the present invention "consists essentially of" a nucleotide sequence if it includes that sequence and additional nucleotides that do not impair the ability of the primer or probe to specifically hybridize to a SARS coronavirus nucleic acid under the conditions selected for performing a diagnostic assay according to the invention.
- SARS coronavirus culture The SARS coronavirus isolate (2003VA2774) , which has been previously sequenced (Ruan et al , 2003), is used for this study.
- the virus stock is propagated in Vero E6 cells (ATCC: C1008) with medium 199 (Sigma Aldrich, USA) supplemented with 5% fetal calf serum (FCS) (Biological Industries, Israel) .
- FCS fetal calf serum
- the culture supernatant is harvested, clarified by centrifugation at 1300 x g, aliquoted and stored at -80°C until use.
- the PFU of the current preparation is determined as 1 x 10(7) PFU/ml .
- Plaque assay A plaque assay is carried out to determine the virus titer in the culture. Briefly, 100 ml series of 10-fold dilution of the virus stock is added to a confluent monolayer of Vero E6 cells in a 24-well plate and incubated for 1 hour at 37°C Following this, 1 ml of 1% carboxymethylcellulose overlay in medium 199 with 5% FCS is then added to each well. After 4 days of incubation at 37°C in 5%C0 2 , the cells are then fixed with 10% formalin and stained with 2% crystal violet. The plaques are counted visually and the virus titer determined. RNA extraction
- SARS coronavirus Standards 10-fold dilution of the stock virus is prepared in serum obtained from a healthy volunteer. RNA is extracted using the QIAGEN Viral RNA Kit (QIAGEN GMbH, Germany) according to the instructions given in the product insert .
- Patient specimens Virus isolation is performed on a bronchoalveolar lavage specimen of SARS cases belonging to the original case cluster from Singapore. RNA is directly extracted from the specimen using a Qiagen QIAamp viral RNA extraction kit (catalog no. 52906) according to the instructions given in the product insert .
- RNA is directly extracted from the stock vial obtained from ATCC (VA, USA) using the QIAGEN Viral RNA Mini Kit (QIAGEN GMbH, Germany) according to the instructions given in the product insert .
- MRC-5 cell line Total RNA is extracted directly from the normal diploid human fibroblast cell line MRC-5 (ATCC CCL171) using a Qiagen RNA extraction kit (catalog no. 74104) and RNA is quantitated using a spectrophotoraeter .
- Example 1 Primer design Primers are selected using; the sequence of the SARS coronavirus Urbani strain (AY2787 1) based on conserved regions of SARS coronavirus genome (Rota et al . 2003 ) .
- a set of primer pairs in the proteinase gene region (position 6652 -7003 , of a non- structural protein 1 (NSPl) region) is found to be most suitable as it exhibits the lowest cross homology with other viruses (Ruan et al . 2003 ) .
- the primers are designed to take into account possible mismatches throughout the genome and to avoid or at least minimize primer dimer formation.
- the NSPl region (proteinase) target of the primers is generally well conserved among i solates of SARS coronavirus .
- NSP9 RNA polymerase
- SARS coronavirus all known isolates
- SARS coronavirus all known isolates
- Table 1 primers targeting this region
- NSTBI BLAST search of all known sequences in the NSTBI database excluding SARS coronavirus sequences .
- Three sets of primer pairs are identified from the NSPl proteinase region. The location of these three sets in relation to the SARS coronavirus genome can be seen in Figure 1.
- the first set amplifies a segment IMCB-1 that is 352 base pairs in length, SEQ ID NO : 2.
- This sequence is flanked by an upper primer (IMCB-l-U, SEQ ID NO: 3) and a lower primer (IMCB-l-L, SEQ ID NO: 4) .
- These primers can be used to specifically detect the presence of SARS coronavirus nucleic acids in a sample.
- IMCB-l-U (19-mer) 5 'ACATCAAATTGCGCTAAGA3 ' (SEQ ID NO: 3)
- IMCB-l-L (21-mer) : 5 'ACAATTCTCTAACGCCATTAC3 ' (SEQ ID NO: 4)
- IMCB-2 of 157 base pairs in length, SEQ ID NO: 5. This sequence is flanked by an upper primer (IMCB-2-U, SEQ ID NO: 6) and a lower primer (IMCB-2 -L, SEQ ID NO : 7) . These primers can be used in like manner to the IMCB-1 primer set to specifically detect the presence of SARS coronavirus nucleic acids in a sample. Both the IMCB-1 and IMCB-2 primer sets can be used with the reagents and conditions set forth, for instance, in the One- Step RT-PCR described in Example 3.
- IMCB-2-U (19-mer) 5 ' GCCGTAGTGTCAGTATCAT3 • (SEQ ID NO: 6)
- IMCB-2-L (20-mer) 5 ' CACCTAACTCTGTACGCTGTC3 ' (SEQ ID NO: 7)
- IMCB-3 a fragment called IMCB-3 that is a portion of IMCB-2 that is 77 base pairs in length, SEQ ID NO: 8. This sequence is flanked by an upper primer (IMCB-3- U, SEQ ID NO: 9) and a lower primer (IMCB-3 -L, SEQ ID NO: 10) .
- a probe oligonucleotide (IMCB-3 -probe, SEQ ID NO: 11) is situated between the two primers of IMCB-3. The primers and the probe are used to detect the presence of SARS coronavirus, for example in a real-time assay using fluorescent detection of the amplified fragment.
- the lower primer of set 2, IMCB-2-L and of set 3, IMCB-3-L, are almost identical, but IMCB-3-L is 3 nucleotides longer at the 3 1 end.
- These primers and/or probe can be used to specifically detect the presence of SARS coronavirus nucleic acids in a sample .
- IMCB-3-U (26-mer) : 5 ' GCACTTTGTAGAAACAGTTTCTTTGG3 ' (SEQ ID NO: 9)
- IMCB- 3 -L (24-mer) : 5 ' CACCTAACTCTGTACGCTGTCCTG3 ' (SEQ ID NO: 10)
- IMCB-3 -Probe (17-mer) 5 ' TGGCTCTTACAGAGATT3 ' (SEQ ID NO: 11)
- Example 2 Detection of SARS coronavirus using the IMCB-1 primer pair
- the first primer pair (IMCB-1) described above is designed for amplifying the portion of the SARS coronavirus genome from nucleotide 6652 to nucleotide 7003.
- the forward primer also herein called an "upper primer” is a 19-mer that hybridizes to the SARS coronavirus genome beginning at position 6652 and has the sequence of SEQ ID NO: 3.
- the reverse primer called herein a “lower primer” is a 21-mer that hybridizes to the SARS coronavirus genome at position 6983 and has the sequence of SEQ ID NO: 4.
- RNA is extracted from samples thought to contain SARS RNA by known methods. The RNA is then converted to DNA using a reverse transcriptase or any other method known in the art. A sample mixture is converted into cDNA in a typical manner using a 1st Strand cDNA Synthesis Kit for RT-PCR (sold by Roche, Basel, Switzerland, catalog no.
- the first strand cDNA reaction is carried out using the following reagents at the indicated concentrations: REAGENTS FINAL CONC . 1. lOx Reaction buffer lx 2. MgCl 2 , 25 mM 5 mM 3. dNTP mix, 10 mM ea. 1 mM ea . 4. Specific primer, 20 ⁇ M 1 ⁇ M (IMCB-1 Lower Primer) 5. RNase inhibitor 50 units 6. AMV reverse transcriptase 20 units 7. Gelatin (0.5 mg/ml) 0.01 mg/ml 8. Sterile water 9. RNA sample
- the reaction is allowed to proceed at 25°C for 10 minutes and then at 42°C for 60 minutes.
- the reverse transcriptase is then inactivated by incubating the reaction at 99°C for 5 minutes and cooling to 4°C for 5 minutes.
- the cDNA is then amplified by adding 1 ⁇ l of 1 ⁇ g/ ⁇ l of single stranded DNA to each sample and preparing the sample for PCR. It will be recognized by those of skill in the art that other methods of amplification known in the art can be performed to amplify the DNA.
- PCR is performed using the following reagents and conditions.
- the reaction mixture is then prepared using the following reagents and concentrations.
- the detection can be alternatively done by an ABI (Foster City, California) PRISM 7000 Sequence Detection System to confirm the presence of the correct amplified region, which is identified as a nucleic acid fragment of 352 nucleotides.
- ABI Anaster City, California
- PRISM 7000 Sequence Detection System to confirm the presence of the correct amplified region, which is identified as a nucleic acid fragment of 352 nucleotides.
- the IMCB-1 primer sets can be used in the Two-Step RT-PCT as well as in the One-Step RT-PCR.
- the IMCB-1 primer sets may be used with the reagents and conditions set forth, for instance, in the One-Step RT-PCR described in Example 3.
- Example 3 SARS coronavirus detection using IMCB-2 primer set
- the second primer pair set described above is designed for amplifying the portion of the SARS coronavirus genome from nucleotide 4609 to nucleotide 4765.
- the forward primer called herein “Upper Primer” or “IMCB-2 -U” is a 19- mer that hybridizes to the SARS coronavirus genomve beginning at position 4609 has the following sequence: IMCB-2-U 5' GCCGTAGTGTCAGTATCAT3 ' (SEQ ID NO: 6)
- the reverse primer, called herein "Primer Lower” or “IMCB-2-L” is a 21-mer that hybridizes to the SARS coronavirus genome at position 4765 has the following sequence : IMCB-2-L 5' CACCTAACTCTGTACGCTGTC3 ' (SEQ ID NO: 7)
- RNA sample is diluted to a 50 ⁇ l reaction volume with 45 ⁇ l of a premixture solution containing reaction buffer, Q-solution (Qiagen, catalog no. 210210) and dNTP mix, at a final concentration of 400 ⁇ M each, and an upper primer and a lower primer at a final concentration of 0.6 ⁇ M each.
- RNase inhibiter at 10 units/ reaction, enzyme mix, RNase free water is also added.
- the thermal cycling is performed using a Stratagene Robocycler 40 (La Jolla, California) with the following steps, reverse transcription at 50°C for 30 min and initial denature at 95° C for 15 min, followed by denaturation at 95° C for 45 sec, annealing at 50°C for 80 sec, extension at 72°C for 50 sec. The cycle is repeated 42 times. The entire RT PCR is completed after a final extension at 72° C for 10 min. The PCR product is analyzed by gel electrophoresis in 2.0% agarose .
- the detection limit of the diagnostic test of the instant invention is approximately 200 copies/ml (1 copy/ 5 ⁇ l reaction) for the virus as confirmed when measured using ARTUS RealArtTM HPA-Coronavirus LC RT PCR Reagents (cat No: 5601-03) .
- Example 4 A RT-PCR SARS diagnostic kit and its use
- a kit according to this Example is typically prepared to contain 50 or 100 reactions.
- the kit is composed of the items listed below; it should be stored at -20°C in a non- frost-free freezer.
- Figure 3C shows results that are obtained using this kit with the IMCB-2 primer set.
- RT-PCR severe acute respiratory syndrome coronavirus
- SARS coronavirus severe acute respiratory syndrome coronavirus
- the region of the SARS coronavirus genome amplified in this assay lies in the the NSP-1 region (proteinase) of the virus genome.
- This kit is optimized to detect a few molecules of the viral RNA in 5 ul of test sample and the entire procedure is performed in one step .
- the kit of this example consists of the following 4 tubes Tube No . Component Storage Conditions
- Tube 1 RT-PCR Enzyme Mix ( e . g . from Qiagen) -20°C, -20°C
- Tube 2 RT-PCR Reaction Mix ( e . g . from Qiagen) 4°C • 20°C
- Tube 3 Primer Mix (Upper & Lower) ( 30 ⁇ M each primer in 20 mM Tris , 1 mM EDTA pH 8 . 2 ) 4°C , 20°C
- Thermal cycling protocol -A This thermal cycling protocol is used for three-block type PCR cyclers such as the RoboCycler ® by Stratagene: Stage Temp (°C) Duration No. Stet Of Cycle (s)
- Thermal cycling protocol -B This thermal cycling condition is for one-block type PCR cycler such as the Px2 Thermal Cycler by Thermo
- thermocycler It is preferred to put mineral oil in the wells of the thermocycler to maximize the conduction of the heat between a reaction tube and a well if it is necessary.
- Example 5 Specificity of RT-PCR using the IMCB primers
- the primer sets designed in Example 1 can be used to detect SARS coronavirus specifically; the amplification of selected viruses is tested by RT-PCR using the IMCB primer sets 1 and 2.
- the following viruses are tested at the indicated titer to check the specificity of the IMCB RT-PCR primer sets 1 and 2 :
- Bovine Coronavirus ATCC VR-874 1.1 x 10(6) PFU/ml
- Table 1 Specificity of IMCB-1 and IMCB-2 primer pairs Sampt ⁇ Cons. 18* ARTUS « ⁇ AR ⁇ US IMCB ⁇ «CB2 MRCBRNA Exl &U U ⁇ iM ⁇ MGUJSive N ⁇ g Neg _fflC5ftMA B « 1 ug/uE t A*m ⁇ nfec»8 ⁇ » ⁇ »w ⁇ «t ⁇ 8s vfttts. NCBI M9Sl6 ⁇ 6.8Xl ⁇
- the Eiken kit is tested using a one step RT-Lamp.
- the IMCB-1 primer pairs are tested using the concentrations of reagents set forth in Example 2. However the reagent materials are those described in Example 3 and the PCR protocol was that of the one step RT-PCR described in Example 3.
- the IMCB-2 primer pairs are tested using the reagent materials and concentrations and the one step RT-PCR protocol described in Example 3.
- the amplification products that are obtained using the IMCB-1 and IMCB-2 primer sets are analysed by agarose gel electrophoresis with ethidium bromide staining.
- the IMCB-3 primer pair is tested using a one step RT-PCR kit prototype optimized for the ABI 7000 Real Time system using a TaqmanTM probe described in Example
- Coronavirus TM RT PCR Abbot List No. B3K360 REV.2003-10-R2. (10 ⁇ l sample is used per reaction instead of 5 ⁇ l) .
- Artus light cycler kit is tested using RealArtTM HPA- Coronavirus LC RT PCR Reagent (Cat No: 5601-03).
- the Roche kit is tested using Light CyclerTM SARS Quantification kit (cat 03604438001) Version 1.
- Table 2 shows the results of the comparative testing.
- the viral sample copy numbers used is decreased from 83 copies per 5 ⁇ l at the top of the table to 0.1 copies per 5 ⁇ l at the bottom of the table in column 1 under the sample volume/run (vol/rxn) .
- the numbers in brackets behind Pos indicate how many positive detections there are per the total number of tests done . From Table 2 it is seen that the IMCB-2 primer sets provide the most sensitive detection; the IMCB- 1 and -2 primer pairs can reliably detect as little as 0 . 8 copies of viral nucleic acid per 5 ⁇ l sample .
- Example 7 Analysis of patient samples Clinical samples are obtained from a number of patients and are analyzed by the assay of the invention, using primer set IMCB-2 and the assay method described in Example 3. The assay method described in Example 4 is used for samples analysed by Artus using their PCR kit . Results are shown in Table 3. Column 1 in Table 3 is a sample identification number. Column 2 is the description of the type of sample that was taken from the patients. Columns 3 and 4 indicate the results of the Artus detection method. Column 5 is the results of the detection using RT PCR with IMCB-2 primer set. The final column 6 documents notes referring to each sample .
- Example 8 SARS Real-Time PCR Diagnostic Kit (RT-PCR)
- RT-PCR Real-Time Reverse Transcriptase- Polymerase Chain Reaction
- SARS coronavirus RNA Severe Acute Respiratory syndrome Coronavirus Ribonucleic Acid
- This kit is optimized for use with the Applied Biosystems Real-Time PCR, ABI Prism 7500, but may be used with other suitable detection platforms as described elsewhere herein.
- the portion of the SARS coronavirus genome amplified by the kit lies in the proteinase region of the SARS coronavirus RNA.
- the kit is sufficiently sensitive to detect a few molecules of RNA in each RT-PCR reaction.
- Tube 1 Reaction Mix (e.g. ABI cat. No. 4309169)
- Tube 2 Enzyme Mix (e.g. ABI cat. No. 4309169)
- Tube 3 Probe Mix (3 ⁇ M upper primer, 3 ⁇ M lower primer, 2 ⁇ M probe in 20 mM Tris, 1 mM EDTA pH 8.2)
- Tube 4 Positive Control (RNA transcripts of the gene targeted by the primers)
- Tube 4 Distilled Water 13.75 ⁇ l RNA Sample 5.0 ⁇ l Total Volume 50.0 ⁇ l Caution should be taken to avoid contamination.
- the IMCB-3 primer set and probe are tested for their ability to detect SARS coronavirus using the Stratagene real-time PCR system Mx3000P.
- the system is used according to the manufacturer's instructions on samples from infected patients. The samples are diluted several fold to a total of viral copy number per 5 ⁇ l ranging between 7.5 to 6. Results are shown in Table 4. The number of viral copies of SARS coronavirus per 5 ⁇ l ranges from 7.5 to 6 in all three runs. Row A (Al-3) is a control no virus sample.
- Row C (C1-C10) is detection of samples at 7.5 viral copies 5 ⁇ l
- Row E (E1-E10) is detection of samples at 7.5 viral copies 5 ⁇ l
- Row G (G1-G10) is detection of samples at 7.5 viral copies 5 ⁇ l .
- the 4 th column indicates how many positive results are detected per number of samples tested. The results demonstrate that the IMCB-3 primer set and probe provide a sensitive and specific assay for SARS coronavirus that is useful in a clinical setting.
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US (1) | US20070248949A1 (en) |
EP (1) | EP1706506A4 (en) |
JP (1) | JP2007514440A (en) |
WO (1) | WO2005059177A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN111635960A (en) * | 2020-05-06 | 2020-09-08 | 温州医科大学附属眼视光医院 | Protective sequence, primer, probe, composition, kit, application and method for steady-state quick-acting detection of novel coronavirus |
CN113512609A (en) * | 2020-09-28 | 2021-10-19 | 上海仁度生物科技股份有限公司 | Novel real-time fluorescent nucleic acid isothermal amplification detection kit for coronavirus, and special primer and probe thereof |
WO2021213163A1 (en) * | 2020-04-21 | 2021-10-28 | The University Of Hong Kong | Identification of nsp1 gene as target of sars-cov-2 real-time rt-pcr using nanopore whole genome sequencing |
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CN111394519B (en) * | 2020-04-13 | 2023-03-21 | 南京实践医学检验有限公司 | Novel coronavirus nucleic acid quantitative detection kit based on digital PCR and application |
US11287396B2 (en) | 2020-06-05 | 2022-03-29 | Princeton Biochemicals, Inc. | Method and system for simultaneous determination of multiple measurable biomarkers during the development of a communicable disease |
CN114752703A (en) * | 2021-01-08 | 2022-07-15 | 苏州绘真生物科技有限公司 | Novel freeze-drying detection reagent for coronavirus nucleic acid and preparation method thereof |
CN113403329B (en) * | 2021-05-17 | 2023-02-28 | 东莞博盛生物科技有限公司 | RNA vaccine for feline coronavirus and construction method thereof |
CN113846186A (en) * | 2021-10-20 | 2021-12-28 | 云南农业大学 | Universal detection primer and detection method for animal beta coronavirus |
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WO2004085455A1 (en) * | 2003-03-24 | 2004-10-07 | The University Of Hong Kong | A diagnostic assay for the human virus causing severe acute respiratory syndrome (sars) |
WO2004099440A1 (en) * | 2003-05-09 | 2004-11-18 | Capital Biochip Company, Ltd. | Methods and compositions for detecting sars virus |
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2004
- 2004-12-17 JP JP2006545303A patent/JP2007514440A/en not_active Withdrawn
- 2004-12-17 WO PCT/SG2004/000416 patent/WO2005059177A1/en active Application Filing
- 2004-12-17 US US10/582,969 patent/US20070248949A1/en not_active Abandoned
- 2004-12-17 EP EP04809236A patent/EP1706506A4/en not_active Withdrawn
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WO2004085455A1 (en) * | 2003-03-24 | 2004-10-07 | The University Of Hong Kong | A diagnostic assay for the human virus causing severe acute respiratory syndrome (sars) |
WO2004099440A1 (en) * | 2003-05-09 | 2004-11-18 | Capital Biochip Company, Ltd. | Methods and compositions for detecting sars virus |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021213163A1 (en) * | 2020-04-21 | 2021-10-28 | The University Of Hong Kong | Identification of nsp1 gene as target of sars-cov-2 real-time rt-pcr using nanopore whole genome sequencing |
CN111635960A (en) * | 2020-05-06 | 2020-09-08 | 温州医科大学附属眼视光医院 | Protective sequence, primer, probe, composition, kit, application and method for steady-state quick-acting detection of novel coronavirus |
CN111635960B (en) * | 2020-05-06 | 2023-06-27 | 温州医科大学附属眼视光医院 | Protective sequence, primer, probe, composition, kit and application and method for steady-state quick-acting detection of novel coronavirus |
CN113512609A (en) * | 2020-09-28 | 2021-10-19 | 上海仁度生物科技股份有限公司 | Novel real-time fluorescent nucleic acid isothermal amplification detection kit for coronavirus, and special primer and probe thereof |
Also Published As
Publication number | Publication date |
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US20070248949A1 (en) | 2007-10-25 |
JP2007514440A (en) | 2007-06-07 |
EP1706506A4 (en) | 2007-10-31 |
EP1706506A1 (en) | 2006-10-04 |
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