CN1258603C - Method and kit for detecting SARS virus in food and animal and plant products - Google Patents
Method and kit for detecting SARS virus in food and animal and plant products Download PDFInfo
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- CN1258603C CN1258603C CN 03148836 CN03148836A CN1258603C CN 1258603 C CN1258603 C CN 1258603C CN 03148836 CN03148836 CN 03148836 CN 03148836 A CN03148836 A CN 03148836A CN 1258603 C CN1258603 C CN 1258603C
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Abstract
The present invention discloses a method and a corresponding detecting kit for detecting SARS viruses in foods, animal products and plant products. The kit comprises a reagent for extracting SARS virus RNA, a reagent for RT-PCR amplification and a reagent for PCR amplification of an SARS virus gene. By simultaneously using two pairs of primers positioned in different conservation areas of an SARS gene and amplifying two specificity fragments of the SARS gene, the amplification specificity is stronger, and simultaneously, the method is highly efficient and timesaving. Thereby, the SARS viruses are effectively detected in the foods, the animal products and the plant products.
Description
Technical field
Present method relates to a kind of method that detects virus composition from food and animals and plants product, particularly a kind of test kit that detects method and this method of SARS virus from food and animals and plants product.
Technical background
Atypical pneumonia is that (Severe Acute Respiratory Syndrome SARS), is that a mutation SARS virus by coronavirus causes to SARS (Severe Acute Respiratory Syndrome).The respiratory disease that SARS virus causes has very strong infectivity, and it is fast to cause a disease, and causes lung failure, threatens people's life.Therefore, from all kinds of samples rapid detection go out SARS virus be the prevention this disease, control the route of transmission an important technology.
At present, the method for inspection to SARS virus mainly contains 3 kinds of antibody test, molecular biology method and cell culture methods etc.Antibody test is to detect by enzyme linked immunological ELISA (IGM/IGA) or immunofluorescence technique clinical symptom antiviral antibody among the SARS patients serum after 20 days to occur, or the M immunoglobulin (Ig) that produces after 10 days of virus infection VERO cell.Molecular detecting method is by to the special pulsating design of primers of SARS virus, utilize RT-PCR, nested PCR method detect the virus comprise in the samples such as blood, ight soil, respiratory secretions (Identification of a Novel Coronavirus inPatients with Severe Acute Respiratory Syndrome C.Drosten and Others " Thenew England Journal of Medicine; Posted April 7; 2003); owing to need successively carry out twice amplification, so required time is longer when adopting nest-type PRC.Cell culture processes is to utilize the VERO cell to detect patient's SARS respiratory secretions and blood sample, whether infected coronavirus with definite patient SARS, but these methods is all at clinical samples.
The method that detects virus in food only has the report of enteroviruses such as comprising hepatitis A virus, poliomyelitis, and the step of extracting enteroviral rna is generally, use the glycine buffer dissolving earlier, precipitate with PEG or other flocculation agent then, then extract RNA with Tri-reagent-poly (dt) magnetic bead.And the Tri-reagent-poly that this method adopted (dt) magnetic bead cost height, and generally be used for low fat product and small sample processing, as the extraction of hepatitis A virus (HAV) RNA in the oyster product.For the check of SARS virus in the food, especially the animal derived food that is polluted in the processing back or the course of processing is not seen relevant report at present as yet.
Summary of the invention
The object of the present invention is to provide a kind of method and relevant detection test kit that from food and animals and plants product, detects SARS virus.This method is applicable to from all kinds of animals and plants products and food samples and detects SARS virus, and it is low to detect cost, and makes detection specificity stronger by 2 conservative region genes of the SARS virus that increases simultaneously, simultaneously high efficiency and time conservation.
The object of the present invention is achieved like this: a kind of method that detects SARS virus from food and animals and plants product has following detection step:
A. the glycine buffer that at first adds the 0.05～0.3M that contains 0.1～0.5M Na salt in food or animals and plants sample makes sample homogenization, in the supernatant liquor of homogenate material after centrifugal, add 10～20% concentration PEG solution that contain 0.3～0.7M Na salt, obtain precipitation, abandon supernatant after centrifugal, add RNA again and extract reagent extraction SARS virus RNA;
B.RT-PCR, the complementary DNA (cDNA) of synthetic RNA;
C. adopt the specificity amplification primer of SARS virus RNA polymerase and N protein gene, by polymerase chain reaction (PCR) amplification RNA polymerase and N protein gene fragment;
D. the amplified production to step (C) carries out electrophoresis detection, and the existence of judging the SARS gene according to electrophoresis result whether.
The preferred PEG8000 of PEG in the steps A; RNA extracts the preferred Trizol reagent of reagent; Preferred RNA reversed transcriptive enzyme is SSII; Preferred SARS virus RNA polymerase specificity amplification primer is F1:5 '-CCTCTCTTGTTCTTGCTCGCA-3 ' and R1:5 '-TATAGTGAGCCGCCACACATG-3 '; The specificity amplification primer of preferred SARS virus N protein gene is F2:5 '-TACACACCTCAGCGTTG-3 ', and R2:5 '-CACGAACGTGACGAAT-3 '.
The invention further relates to a kind of test kit that detects SARS virus from food, animals and plants and products thereof, this test kit comprises following component:
(a) SARS virus RNA extracts reagent: comprise solution A: the glycine buffer that contains 0.05～0.3M of 0.1～0.5M Na salt; Solution B: 10～20% concentration PEG solution that contain 0.3～0.7M Na salt; Solution C: RNA extracts reagent; And solution D: DEPC treating water;
(b) RT-PCR amplifing reagent: primer OligdT, dNTP solution, DTT and RNA reversed transcriptive enzyme;
(c) reagent of pcr amplification SARS virus gene: comprise the specificity amplification primer of SARS virus RNA polymerase and N protein gene, the reagent that archaeal dna polymerase and pcr amplification are required.The preferred PEG8000 of the PEG in the solution B wherein; The preferred Trizol reagent of solution C; Preferred RNA reversed transcriptive enzyme is SSII; Preferred SARS virus RNA polymerase specificity amplification primer is F1:5 '-CCTCTCTTGTTCTTGCTCGCA-3 ' and R1:5 '-TATAGTGAGCCGCCACACATG-3 '; The specificity amplification primer of preferred SARS virus N protein gene is F2:5'-TACACACCTCAGCGTTG-3 ', and R2:5 '-CACGAACGTGACGAAT-3 '; Preferred archaeal dna polymerase is the Taq enzyme.
" reagent that pcr amplification is required " is meant that the gene with the denier in the template carries out the used reagent of pcr amplification reaction process, comprises dNTP, 10 * enzyme buffer liquid and distilled water etc.
Viral RNA extracting method of the present invention: glycine buffer-PEG precipitation-Trizol extracts, can be effectively from all kinds of sample, and comprise in the animal source sample of lipid and extract SARS virus RNA, and this method cost is low.By using two pairs of primers being in the different conservative regions of SARS gene simultaneously, 2 the SASR gene specific fragments that increases make the specificity of amplification stronger among the present invention, the while high efficiency and time conservation, thus reach purpose to the SARS gene test.
Description of drawings
Figure 1A is the detection of SARS virus in the chicken tissue
Figure 1B is the detection of SARS virus in the chicken tissue
Fig. 2 A is the detection of SARS virus in the aquatic products tissue
Fig. 2 B is the detection of SARS virus in the aquatic products tissue
Fig. 3 A is the detection of SARS virus in the vegetables
Fig. 3 B is the detection of SARS virus in the vegetables
Embodiment
The solution C of the RNA of SARS virus extraction reagent is a Trizol reagent among the embodiment.Concrete component among the embodiment in the RT-PCR amplifing reagent, and the concrete component in the reagent of pcr amplification SARS virus gene is as described in the following table.
The RT-PCR amplifing reagent
| The component title | Specification (μ l) | The |
| Primer | ||
| 1 | 50 | 0.5μg/μl |
| Reaction mixture | ||
| 1 | 175 | 5 * Buffer (87 μ l), 10mM dNTP mixture (44 μ l), 100mM DTT (44 μ l) |
| | 20 | 200u/ μ l reversed transcriptive enzyme SSII |
The reagent of pcr amplification SARS virus gene
| The component title | Specification (μ l) | The reagent composition |
| Reaction mixture 2.1 | 1000 | 10 * PCR Buffer (128 μ l), 10mM dNTP (26 μ l), 10 μ M primers F 1 (26 μ l), 10 μ M primer R1 (26 μ l), dd H 2O(794μl) |
| Reaction mixture 2.2 | 1000 | 10 * PCR Buffer (128 μ l), 10mM dNTP (26 μ l), 10 μ M primers F 2 (26 μ l), 10 μ M primer R2 (26 μ l), dd H 2O(794μl) |
| | 40 | 2u/μl Taq |
Take by weighing 25g chicken sample, (pH9.5), homogeneous is 5 times in tissue mashing machine for 0.1M glycine, 0.3MNaCl, and each 45s makes its abundant homogenate to add 175ml 1 * solution A.Left standstill 5 minutes; Get the PV centrifuge tube of 50ml band screw-cap, add 30ml homogenate material, 12000rpm, 4 ℃ are centrifugal 15 minutes; Supernatant is transferred in the new PV centrifuge tube, added isopyknic solution B (20%PEG8000,0.7M NaCl), placed on ice 1 hour, 9000rpm, 4 ℃ are centrifugal 15 minutes; Abandon supernatant, every pipe adds 5ml solution C (Trizol), and piping and druming suspends, and room temperature left standstill 5 minutes; Add the 1ml chloroform, concussion shakes up repeatedly, and room temperature left standstill 10 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm carefully draw the upper strata water in the 1.5ml Eppendorf tube that does not contain the RNA enzyme, add the Virahol of 0.5 times of volume, and room temperature left standstill 10 minutes, 12000rpm, and 4 ℃ are centrifugal 10 minutes; Abandon supernatant, wash 2 times with 75% ethanol of precooling 500 μ l, the careful suction abandoned supernatant, dries or 90 ℃ of flash bakings, needs 5-10 minute approximately; Add 10 μ l DEPC treating water dissolvings RNA, mixing gently, it is synthetic or be stored in-70 ℃ to be used for cDNA immediately.
Get in RNA to new Eppendorf tube that does not contain the RNA enzyme of 10 μ l, add 2 μ l primers 1 (0.5 μ g/ μ l OligdT), place 70 ℃ of water-baths, reacted 10 minutes, placed immediately ice bath 2-5 minute; The reaction mixture 1 that adds 7 μ l, mixing is of short duration centrifugal gently, and room temperature was placed 5 minutes; 1,42 ℃ of the enzyme mixture 60 minutes that adds 1 μ l, 70 ℃ 15 minutes, placed immediately ice bath 2-5 minute.
Get the Eppendorf tube of two PCR reactions, mark, each adds the reaction product in the operation steps two of 10 μ l, the reaction mixture 2.1 and the reaction mixture 2.2 that add 39 μ l more respectively, the enzyme mixture 2.2 that respectively adds 1 μ l respectively adds the enzyme mixture 2 (2u/ μ l Taq) of 1 μ l again, and is of short duration centrifugal, add two drop of liquid paraffin (the PCR instrument with the lid heating-type can not add), place on the PCR instrument and react; The setting of PCR reaction parameter
Pre-sex change: 95 ℃ 120 seconds
Extend eventually: 72 ℃ 300 seconds
The product of step 4, electrophoresis detection pcr amplification
Get the 2.0g agarose, in the heating of 100ml electrophoretic buffer, fully dissolving adds ethidium bromide to final concentration 1 μ g/ml, glue.In electrophoresis chamber, add an amount of electrophoretic buffer, 10 μ l pcr amplification products are mixed point sample respectively at an amount of sample loading buffer.The 9V/cm constant voltage, electrophoresis 20-30min.Observations and write down result's (seeing Figure 1A and Figure 1B) under the Ultraviolet Detector.
Wherein swimming lane 1 is molecular weight Marker; Swimming lane 2 has added the electrophoresis of chicken meat sample after above-mentioned same steps as of 100TCID50/ml SARS virus for adopting, the result is positive, wherein Figure 1A has detected SARS virus rna polymerase gene (seeing 121bp arrow indication place among Figure 1A), has detected N protein gene (seeing 182bp arrow indication place among Figure 1B) among Figure 1B; Swimming lane 3-7 is that present embodiment is with a check sample repeated experiments, negative result.
Take by weighing 20g razor clam clam tissue, (pH8.5), homogeneous is 5 times in tissue mashing machine for 0.3M glycine, 0.5MNaCl, and each 45s makes its abundant homogenate to add the 150ml solution A.Left standstill 5 minutes; Get the PV centrifuge tube of 50ml band screw-cap, add 30ml homogenate material, 12000rpm, 4 ℃ are centrifugal 15 minutes; Supernatant is transferred in the new PV centrifuge tube, add isopyknic solution B (14%PEG8000 0.4MNaCl), placed 1 hour on ice, 9000rpm, 4 ℃ are centrifugal 10 minutes; Abandon supernatant, every pipe adds the 5ml solution C, and piping and druming suspends, and room temperature left standstill 5 minutes; Add the 1ml chloroform, concussion shakes up repeatedly, and room temperature left standstill 10 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm carefully draw the upper strata water in the 1.5ml Eppendorf tube that does not contain the RNA enzyme, add the Virahol of 0.5 times of volume, and room temperature left standstill 10 minutes, 12000rpm, and 4 ℃ are centrifugal 10 minutes; Abandon supernatant, wash 2 times with 75% ethanol of precooling 500 μ l, the careful suction abandoned supernatant, dries or 90 ℃ of flash bakings, needs 5-10 minute approximately; Add 10 μ l DEPC treating water dissolving RNA, mixing is used for cDNA immediately and synthesizes gently.
Get in the new Eppendorf tube that does not contain the RNA enzyme of 10 μ l RNA to, add 2 μ l primers 1, place 70 ℃ of water-baths, reacted 10 minutes, placed immediately ice bath 2-5 minute; The reaction mixture 1 that adds 7 μ l, mixing is of short duration centrifugal gently, and room temperature was placed 5 minutes; 1,42 ℃ of the enzyme mixture 60 minutes that adds 1 μ l, 70 ℃ 15 minutes, placed immediately ice bath 2-5 minute.
Get the Eppendorf tube of two PCR reactions, mark, each adds the reaction product in the operation steps two of 10 μ l, the reaction mixture 2.1 and the reaction mixture 2.2 that add 39 μ l more respectively, the enzyme mixture 2 that respectively adds 1 μ l again, of short duration centrifugal, add two drop of liquid paraffin (the PCR instrument with the lid heating-type can not add), place on the PCR instrument and react; The setting of PCR reaction parameter
Pre-sex change: 95 ℃ 120 seconds
Extend eventually: 72 ℃ 300 seconds
The product of step 4, electrophoresis detection pcr amplification
Get the 2.0g agarose.In the heating of 100ml electrophoretic buffer, fully dissolving adds ethidium bromide to final concentration 1 μ g/ml, glue.In electrophoresis chamber, add an amount of electrophoretic buffer, 10 μ l pcr amplification products are mixed point sample respectively at an amount of sample loading buffer.The 9V/cm constant voltage, electrophoresis 20-30min.Observations and write down result's (seeing Fig. 2 A and Fig. 2 B) under the Ultraviolet Detector.The electrophoresis of wherein swimming lane 2 positive contrasts (having added the 10TCID50/ml SARS virus in the sample) after above-mentioned same steps as, the result is positive.Swimming lane 3-7 is that present embodiment is with a check sample repeated experiments, negative result.
Embodiment 3: detect SARS virus from vegetable sample
Take by weighing the 25g vegetable sample, (pH8.5), homogeneous is 5 times in tissue mashing machine for 0.05M glycine, 0.1MNaCl, and each 45s makes its abundant homogenate to add the 170ml solution A.Left standstill 5 minutes; Get the PV centrifuge tube of 50ml band screw-cap, add 30ml homogenate material, 12000rpm, 4 ℃ are centrifugal 15 minutes; Supernatant is transferred in the new PV centrifuge tube, added isopyknic solution B (10%PEG8000,0.3M NaCl), placed on ice 1 hour, 9000rpm, 4 ℃ are centrifugal 10 minutes; Abandon supernatant, every pipe adds the 5ml solution C, and piping and druming suspends, and room temperature left standstill 5 minutes; Add the 1ml chloroform, concussion shakes up repeatedly, and room temperature left standstill 10 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm carefully draw the upper strata water in the 1.5ml Eppendorf tube that does not contain the RNA enzyme, add the Virahol of 0.5 times of volume, and room temperature left standstill 10 minutes, 12000rpm, and 4 ℃ are centrifugal 10 minutes; Abandon supernatant, wash 2 times with 75% ethanol of precooling 500 μ l, the careful suction abandoned supernatant, dries or 90 ℃ of flash bakings, needs 5-10 minute approximately; Add 10 μ l DEPC treating water dissolving RNA, mixing is used for cDNA immediately and synthesizes gently.
Get in the new Eppendorf tube that does not contain the RNA enzyme of 10 μ l RNA to, add 2 μ l primers 1, place 70 ℃ of water-baths, reacted 10 minutes, placed immediately ice bath 2-5 minute; The reaction mixture 1 that adds 7 μ l, mixing is of short duration centrifugal gently, and room temperature was placed 5 minutes; 1,42 ℃ of the enzyme mixture 60 minutes that adds 1 μ l, 70 ℃ 15 minutes, placed immediately ice bath 2-5 minute.
Get the Eppendorf tube of two PCR reactions, mark, each adds the reaction product in the operation steps two of 10 μ l, the reaction mixture 2.1 and the reaction mixture 2.2 that add 39 μ l more respectively, the enzyme mixture 2 that respectively adds 1 μ l again, of short duration centrifugal, add two drop of liquid paraffin (the PCR instrument with the lid heating-type can not add), place on the PCR instrument and react; The setting of PCR reaction parameter
Pre-sex change: 95 ℃ 120 seconds
Extend eventually: 72 ℃ 300 seconds
The product of step 4, electrophoresis detection pcr amplification
Get the 2.0g agarose.In the heating of 100ml electrophoretic buffer, fully dissolving adds ethidium bromide to final concentration 1 μ g/ml, glue.In electrophoresis chamber, add an amount of electrophoretic buffer, 10 μ l pcr amplification products are mixed point sample respectively at an amount of sample loading buffer.The 9V/cm constant voltage, electrophoresis 20-30min.Observations and write down result's (seeing Fig. 3 A and Fig. 3 B) under the Ultraviolet Detector.The electrophoresis of wherein swimming lane 2 positive contrasts (having added the vegetables of 10TCID50/mlSARS virus) after above-mentioned same steps as, the result is positive.Swimming lane 3-7 is that present embodiment is with a check sample repeated experiments, negative result.
Claims (10)
1. method that detects SARS virus from food and animals and plants product has following detection step:
A. the glycine buffer that at first adds the 0.05～0.3M that contains 0.1～0.5M Na salt in food or animals and plants sample makes sample homogenization, in the supernatant liquor of homogenate material after centrifugal, add 10～20% concentration PEG solution that contain 0.3～0.7M Na salt, obtain precipitation, abandon supernatant after centrifugal, add RNA again and extract reagent extraction SARS virus RNA;
B. RT-PCR then is with the complementary DNA (cDNA) of the synthetic RNA of RNA reversed transcriptive enzyme;
C. adopt the specificity amplification primer of SARS virus RNA polymerase and N protein gene, by polymerase chain reaction (PCR) amplification RNA polymerase and N protein gene;
D. the amplified production to step (C) carries out electrophoresis detection, and the existence of judging the SARS gene according to electrophoresis result whether.
2. method according to claim 1 is characterized in that, the PEG in the steps A is PEG8000.
3. method according to claim 1 is characterized in that, it is Trizol reagent that the RNA in the steps A extracts reagent.
4. method according to claim 1 is characterized in that, the RNA reversed transcriptive enzyme among the step B is SSII.
5. method according to claim 1 is characterized in that, the SARS virus RNA polymerase specificity amplification primer among the step C is F1:5 '-CCTCTCTTGTTCTTGCTCGCA-3 ' and R1:5 '-TATAGTGAGCCGCCACACATG-3 '.
6. method according to claim 1 is characterized in that, the specificity amplification primer of the SARS virus N protein gene among the step C is F2:5 '-TACACACCTCAGCGTTG-3 ', and R2:5 '-CACGAACGTGACGAAT-3 '.
7. test kit that from food and animals and plants product, detects SARS virus, this test kit comprises following component:
(a) SARS virus RNA extracts reagent: comprise solution A: the glycine buffer that contains 0.05～0.3M of 0.1～0.5M Na salt; Solution B: the PEG solution that contains 10～20% concentration of 0.3～0.7M Na salt; Solution C: RNA extracts reagent; And solution D: DEPC treating water;
(b) RT-PCR amplifing reagent: primer OligdT, dNTP solution, DTT and RNA reversed transcriptive enzyme;
(c) reagent of pcr amplification SARS virus gene: comprise the specificity amplification primer of SARS virus RNA polymerase and N protein gene, the reagent that archaeal dna polymerase and pcr amplification are required.
8. the test kit of detection SARS virus according to claim 7 is characterized in that, the PEG in the solution B is PEG8000.
9. the test kit of detection SARS virus according to claim 7 is characterized in that, it is Trizol reagent that the RNA in the solution C extracts reagent.
10. the test kit of detection SARS virus according to claim 7 is characterized in that, the RNA reversed transcriptive enzyme in the component (b) is SSII.
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| CN 03148836 CN1258603C (en) | 2003-06-13 | 2003-06-13 | Method and kit for detecting SARS virus in food and animal and plant products |
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| CN 03148836 CN1258603C (en) | 2003-06-13 | 2003-06-13 | Method and kit for detecting SARS virus in food and animal and plant products |
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| CN1258603C true CN1258603C (en) | 2006-06-07 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11361847B1 (en) | 2021-02-06 | 2022-06-14 | Timothy A. Hodge | System and method for rapidly reporting testing results |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11361847B1 (en) | 2021-02-06 | 2022-06-14 | Timothy A. Hodge | System and method for rapidly reporting testing results |
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