CN111323512B - Kit for detecting inactivated virus and detection method - Google Patents
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Abstract
The invention discloses a kit for detecting inactivated viruses, which comprises the following components: (1) buffer solution, (2) reducing agent, (3) alkylating reagent, (4) trypsin, (5) stop solution, (6) heat-stable standard polypeptide; the amino acid sequence of the thermostable standard polypeptide is as follows: LPLGINITNFR are provided. The invention can detect the inactivated virus and obtain an accurate detection result on the premise of ensuring the sensitivity and specificity of detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a kit and a detection method for detecting inactivated viruses.
Background
Viruses are a class of non-cellular microorganisms. Mature intact virus particles become virions, composed of nucleic acids and proteins. The virus is characterized by small volume and can pass through a sterilizing filter; simple structure, only contains one type of nucleic acid (DNA or RNA); lacking genes encoding mitochondria and ribosomes, it is necessary to parasitize living cells and reproduce progeny in a replicative manner.
Since 1892 for the first discovery of tobacco mosaic virus, more than four thousand animal and plant viruses have been discovered so far, of which more than 500 are pathogenic to human. In clinical microbial infection, the virus causes about 75 percent of the infection, and some infectious guns have the advantages of rapid transmission, wide prevalence, high mortality rate and serious sequelae.
In virus clinical testing methods, gene (DNA or RNA) based methods (e.g., PCR or sequencing) and protein fingerprint based methods (e.g., time-of-flight mass spectrometry) are commonly used. They all require the collection of fresh samples (pharyngeal swabs, sputum, lung perfusate or blood) from patients for testing, but these samples can still pose a significant risk of infection to health care and testing personnel. The collected sample is immediately inactivated, so that the infection risk of medical staff and detection staff can be effectively reduced, but after the sample is inactivated (an autoclave is usually adopted), although the virus loses the original pathogenicity, detection markers (DNA, RNA and protein with a spatial structure) of the virus are easy to damage and cannot be detected. Therefore, how to effectively reduce the infection risk of medical staff and detection staff, and meanwhile, the detection is not influenced, which becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a kit for detecting inactivated viruses, which can detect the inactivated viruses to obtain accurate detection results on the premise of ensuring the sensitivity and specificity of detection.
The invention also provides a virus detection method for reducing the infection risk of medical personnel, which can detect the inactivated virus to obtain an accurate detection result and effectively reduce the infection risk of the medical personnel and the detection personnel.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a kit for detection of inactivated virus comprising the following components: (1) buffer solution, (2) reducing agent, (3) alkylating reagent, (4) trypsin, (5) stop solution, (6) heat-stable standard polypeptide; the amino acid sequence of the thermostable standard polypeptide is as follows: LPLGINITNFR are provided.
The invention abandons the traditional and easily-damaged detection markers (DNA, RNA and protein with a spatial structure), and selects the primary structure sequence of the protein/polypeptide with stable chemical properties as the marker. Suitable amino acid sequences are identified and screened for each virus as markers, and the screened markers are detected by liquid chromatography mass spectrometry. The liquid chromatography tandem mass spectrometry is a device for identifying the molecular weight of a detected object, and has higher sensitivity and accuracy. The amino acid sequence is one of the materials which embody the genetic code in organisms, and the specific amino acid sequence can be selected to ensure the specificity of detection.
Compared with the spatial structure of DNA, RNA and protein, the amino acid sequence has higher chemical stability. Compared with the traditional classical method, the invention greatly improves the stability of detection on the premise of ensuring the sensitivity and specificity of detection. The inactivated virus can be detected, and the sample can be detected even in an extremely environment without affecting the detection result.
The buffer solution is ammonium bicarbonate solution with the concentration of 50-1000 mmol/L.
The reducing agent is dithiothreitol solution with the concentration of 20-200 mmol/L.
The alkylating reagent is iodoacetamide solution with the concentration of 60-600 mmol/L.
The trypsin is alkaline trypsin with the concentration of 100-1000 mg/L.
The stop solution is formic acid with the mass concentration of 60-98%.
The concentration of the thermostable standard polypeptide is 20-1000 ng/ml.
A non-medical virus detection method for reducing the risk of infection of a medical worker, comprising the steps of:
(1) adopting a liquid chromatography tandem mass spectrum as a detection instrument, taking a thermostable standard polypeptide as a standard substance, carrying out sample injection analysis, and establishing a standard map;
(2) directly carrying out damp-heat sterilization on the collected sample, wherein the damp-heat sterilization condition is that the sample is sterilized for 30 minutes at 121 ℃ in a high-pressure steam sterilizer;
(3) carrying out enzymolysis on the sterilized sample by using components of the kits (1) - (5);
(4) and (4) detecting the sample subjected to enzymolysis by using a liquid chromatography tandem mass spectrum.
The purpose of this scheme is compromise under the prerequisite of accurate testing result, reduces medical personnel and testing personnel's infection risk as far as, makes its infectivity go out through the sample direct sterilization that will gather, realizes reducing medical personnel and testing personnel's infection risk. Because the specific amino acid marker is adopted, the method is not influenced by sterilization operation, the medical workers can be protected from the risk of microbial infection on the premise of obtaining an accurate detection result, and meanwhile, a laboratory with a lower biological safety level can also accept detection work. The purpose of the scheme is not to judge the health condition of the detected object, and the scheme is only limited to the improvement of the detection program so as to reduce the infection risk of medical staff and detection personnel as far as possible and prevent the medical staff and the detection personnel from being infected by viruses due to the detected object.
The step (3) is specifically as follows: and (3) adding 0.665mL buffer solution and 10 muL reducing agent into 500 muL of the sterilized sample to be detected, reducing the sample in a water bath at 50 ℃ for 30 minutes, then adding 10 muL alkylating reagent, standing the sample in a dark place at room temperature for 30 minutes, adding 10 muL trypsin, carrying out enzymolysis in a water bath at 37 ℃ overnight, and finally adding 5 muL stop solution to stop the enzymolysis reaction.
The detection conditions of the liquid chromatogram tandem mass spectrum are as follows:
the liquid phase conditions were:
a chromatographic column: waters BEH 300C18, specification 100mm × 2.1mm, particle size 1.7 μm;
column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 3% to 32% in 5 minutes;
flow rate: 0.3 mL/min;
sample introduction volume: 5 mu L of the solution;
the mass spectrum detection conditions are as follows:
ESI + ion source, capillary voltage: 3.0kv, cone voltage: 35V, desolventizing temperature: 500 ℃, desolventizing gas flow: 900L/min, cone hole back-blowing gas flow: 150L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high end pointResolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 150 ℃, extractor voltage: 3.0V, the monitoring mode adopts a multi-reaction detection mode MRM, and the concentration of parent ions: 1257.7, daughter ion: 764.4, 650.4.
The invention has the beneficial effects that:
1. on the premise of ensuring the sensitivity and specificity of detection, the inactivated virus can be detected to obtain an accurate detection result.
2. The detection process is improved to reduce the infection risk of medical staff and detection staff as much as possible and prevent the medical staff and the detection staff from being infected by virus due to the detected object.
Drawings
FIG. 1 is a graph of the thermostability of different polypeptides.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1:
a kit for detection of inactivated virus comprising the following components: (1) buffer solution: ammonium bicarbonate solution with concentration of 50mmol/L, (2) reducing agent: dithiothreitol solution at a concentration of 20mmol/L, (3) alkylating reagent: iodoacetamide solution at a concentration of 60mmol/L, (4) trypsin: alkaline trypsin at a concentration of 100mg/L, (5) stop solution: formic acid with the mass concentration of 60 percent, (6) thermostable standard polypeptide; the amino acid sequence of the thermostable standard polypeptide is as follows: LPLGINITNFR (SEQ ID No.1), at a concentration of 20 ng/ml.
Example 2:
a kit for detection of inactivated virus comprising the following components: (1) buffer solution: ammonium bicarbonate solution with concentration of 1000mmol/L, (2) reducing agent: dithiothreitol solution with concentration of 200mmol/L, (3) alkylating reagent: iodoacetamide solution at a concentration of 600mmol/L, (4) trypsin: alkaline trypsin at a concentration of 1000mg/L, (5) stop solution: formic acid with mass concentration of 98 percent, (6) thermostable standard polypeptide; the amino acid sequence of the thermostable standard polypeptide is as follows: LPLGINITNFR (SEQ ID No.1), at a concentration of 1000 ng/ml.
Example 3:
a kit for detection of inactivated virus comprising the following components: (1) buffer solution: ammonium bicarbonate solution with concentration of 500mmol/L, (2) reducing agent: dithiothreitol solution with concentration of 100mmol/L, (3) alkylating reagent: iodoacetamide solution at a concentration of 300mmol/L, (4) trypsin: alkaline trypsin at a concentration of 500mg/L, (5) stop solution: formic acid with mass concentration of 98 percent, (6) thermostable standard polypeptide; the amino acid sequence of the thermostable standard polypeptide is as follows: LPLGINITNFR (SEQ ID No.1), at a concentration of 300 ng/ml.
Example 4:
a non-medical virus detection method for reducing the risk of infection of a medical worker, comprising the steps of:
(1) adopting liquid chromatography tandem mass spectrometry as a detection instrument, taking thermostable standard polypeptide as a standard substance (the concentration is 300ng/ml), carrying out sample injection analysis, and establishing a standard map;
(2) directly carrying out damp-heat sterilization on the collected sample, wherein the damp-heat sterilization condition is that the sample is sterilized for 30 minutes at 121 ℃ in a high-pressure steam sterilizer;
(3) taking 500 mu L of a sterilized sample to be detected, adding 0.665mL of buffer solution and 10 mu L of reducing agent, reducing in a water bath at 50 ℃ for 30 minutes, then adding 10 mu L of alkylating reagent, standing in a dark place at room temperature for 30 minutes, adding 10 mu L of trypsin, carrying out enzymolysis in a water bath at 37 ℃ overnight, finally adding 5 mu L of stop solution, and stopping the enzymolysis reaction;
(4) and (4) detecting the sample subjected to enzymolysis by using a liquid chromatography tandem mass spectrum.
The detection conditions of the liquid chromatogram tandem mass spectrum are as follows:
the liquid phase conditions were:
a chromatographic column: waters BEH 300C18, specification 100mm × 2.1mm, particle size 1.7 μm;
column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 3% to 32% in 5 minutes;
flow rate: 0.3 mL/min;
sample introduction volume: 5 μ L.
The mass spectrum detection conditions are as follows:
ESI + ion source, capillary voltage: 3.0kv, cone voltage: 35V, desolventizing temperature: 500 ℃, desolventizing gas flow: 900L/min, cone hole back-blowing gas flow: 150L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 150 ℃, extractor voltage: 3.0V, and a multi-reaction detection mode MRM is adopted as a monitoring mode.
MRM parameters are as follows
The present invention is described by taking a novel coronavirus (SARS-CoV-2, positive sample collected by the hospital itself) as an example.
Example 1: inactivation identification method for novel coronavirus in alveolar lavage fluid
1. Sterilizing alveolar perfusate of a patient at 121 ℃ for 30 minutes;
2. taking 500 mu L of the sterilized perfusate, adding 0.665mL ammonium bicarbonate solution (500mmol/L) and 10 mu L dithiothreitol solution (100mmol/L), reducing in a water bath at 50 ℃ for 30 minutes, then adding 10 mu L iodoacetamide solution (300mmol/L), standing at room temperature in the dark for 30 minutes, adding 10 mu L trypsin (500mg/L), carrying out enzymolysis in a water bath at 37 ℃ overnight, finally adding 5 mu L concentrated formic acid (98%), and stopping the enzymolysis reaction.
3. The enzymolysis product is detected by liquid chromatography-tandem mass spectrometry under the same detection conditions as in example 4.
Example 2: identification of specificity
To verify the specificity of the assay of the present invention, lung perfusate from patients with influenza a, influenza b and novel coronavirus was selected as a sample (two replicates per sample) for testing as described in example 1.
| Sample numbering | To confirm the diagnosis of the disease | The result of the detection |
| 1 | Influenza A | Negative of |
| 2 | Influenza A | Negative of |
| 3 | Influenza B | Negative of |
| 4 | Influenza B | Negative of |
| 5 | Novel coronavirus | Positive for |
| 6 | Novel coronavirus | Positive for |
The detection result shows that the method has specificity and can be effectively distinguished from other viruses. Compared with the commercialized novel coronavirus nucleic acid detection kit used in the hospital, the commercialized kit detects a sterilized sample, and the two detection results are negative and cannot be detected.
Sources of thermostable standard polypeptides: screening of polypeptides for thermostability
The method has the greatest advantage that the sample after high-temperature sterilization can be detected. Therefore, the stability of the amino acid sequence needs to be rigorously examined to determine whether it is suitable for clinical examination.
The research selects coronavirus S protein as a research target, 78 polypeptides can be theoretically generated by enzymolysis, and 34 polypeptides are actually obtained by identification. After the 34 polypeptide detection methods are established on a liquid chromatography tandem mass spectrum, 10 polypeptides with the highest sensitivity are selected for further research. Polypeptide list see Table below
| Numbering | Polypeptide sequence |
| A | FDEDDSEPVLK(SEQ ID No.2) |
| B | GVYYPDEIFR(SEQ ID No.3) |
| C | LPLGINITNFR(SEQ ID No.1) |
| D | GWVFGSTMNNK(SEQ ID No.4) |
| E | SFIEDLLFNK(SEQ ID No.5) |
| F | FQPFQQFGR(SEQ ID No.6) |
| G | FPSVYAWER(SEQ ID No.7) |
| H | DVSDFTDSVR(SEQ ID No.8) |
| I | ALSGIAAEQDR(SEQ ID No.9) |
| J | DGIYFAATEK(SEQ ID No.10) |
。
One portion of the positive alveolar perfusate was taken as a sample, divided into 7 portions, and autoclaved at 121 ℃ for 0, 15, 30, 45, 60, 90, and 120 minutes, respectively. And then detecting by adopting liquid chromatography tandem mass spectrometry. Recovery of the samples was calculated at several sterilization times with 0 min (i.e. samples not subjected to high temperature sterilization) as 100% recovery. Wherein 3 polypeptides still provide a recovery rate of more than 75% under a sterilization time of 30 minutes, and are suitable as candidate markers. Among them, the polypeptide having the sequence of LPLGINITNFR (FIG. 1) is most thermostable and is finally used as a marker of the present invention.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
SEQUENCE LISTING
<110> Zhejiang university medical college affiliated with the fourth hospital (Zhejiang Uygur Hospital, Zhejiang university medical college affiliated with the fourth hospital)
<120> kit for detecting inactivated virus and detection method
<130> 2020.03
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 11
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 1
Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg
1 5 10
<210> 2
<211> 11
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 2
Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys
1 5 10
<210> 3
<211> 10
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 3
Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg
1 5 10
<210> 4
<211> 11
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 4
Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys
1 5 10
<210> 5
<211> 10
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 5
Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys
1 5 10
<210> 6
<211> 9
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 6
Phe Gln Pro Phe Gln Gln Phe Gly Arg
1 5
<210> 7
<211> 9
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 7
Phe Pro Ser Val Tyr Ala Trp Glu Arg
1 5
<210> 8
<211> 10
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 8
Asp Val Ser Asp Phe Thr Asp Ser Val Arg
1 5 10
<210> 9
<211> 11
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 9
Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg
1 5 10
<210> 10
<211> 10
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 10
Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys
1 5 10
Claims (6)
1. A kit for detecting inactivated viruses, comprising the following components: (1) buffer solution, (2) reducing agent, (3) alkylating reagent, (4) trypsin, (5) stop solution, (6) heat-stable standard polypeptide; the amino acid sequence of the thermostable standard polypeptide is as follows: LPLGINITNFR, respectively;
the buffer solution is an ammonium bicarbonate solution with the concentration of 50-1000 mmol/L; the reducing agent is a dithiothreitol solution with the concentration of 20-200 mmol/L; the alkylating reagent is iodoacetamide solution with the concentration of 60-600 mmol/L; the stop solution is formic acid with the mass concentration of 60-98%; the virus is a novel coronavirus SARS-CoV-2.
2. The kit for detecting an inactivated virus according to claim 1, wherein: the trypsin is alkaline trypsin with the concentration of 100-1000 mg/L.
3. The kit for detecting an inactivated virus according to claim 1, wherein: the concentration of the thermostable standard polypeptide is 20-1000 ng/ml.
4. A non-medical virus detection method for reducing the risk of infection of a healthcare worker, comprising the steps of:
(1) adopting a liquid chromatography tandem mass spectrum as a detection instrument, taking a thermostable standard polypeptide as a standard substance, carrying out sample injection analysis, and establishing a standard map;
(2) directly carrying out damp-heat sterilization on the collected sample, wherein the damp-heat sterilization condition is that the sample is sterilized for 30 minutes at 121 ℃ in a high-pressure steam sterilizer;
(3) carrying out enzymolysis on a sterilized sample by adopting the components of the kits (1) - (5) of claim 1;
(4) detecting the sample after enzymolysis by liquid chromatography-tandem mass spectrometry;
the virus is a novel coronavirus SARS-CoV-2, and the amino acid sequence of the thermal stability standard polypeptide is as follows: LPLGINITNFR are provided.
5. The method of claim 4, wherein: the step (3) is specifically as follows: and adding 0.665mL of buffer solution and 10 mu L of reducing agent into 500 mu L of sterilized sample to be detected, reducing the sample in water bath at 50 ℃ for 30 minutes, then adding 10 mu L of alkylating reagent, standing the sample in a dark place at room temperature for 30 minutes, adding 10 mu L of trypsin, carrying out enzymolysis in water bath at 37 ℃ overnight, and finally adding 5 mu L of stop solution to stop the enzymolysis reaction.
6. The method of claim 4, wherein: the detection conditions of the liquid chromatogram tandem mass spectrum are as follows:
the liquid phase conditions were:
a chromatographic column: waters BEH 300C18, specification 100mm × 2.1mm, particle size 1.7 μm;
column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 3% to 32% in 5 minutes;
flow rate: 0.3 mL/min;
sample introduction volume: 5 mu L of the solution;
the mass spectrum detection conditions are as follows:
ESI + ion source, capillary voltage: 3.0kv, cone voltage: 35V, desolventizing temperature: 500 ℃, desolventizing gas flow: 900L/min, cone hole back-blowing gas flow: 150L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1:2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 150 ℃, extractor voltage: 3.0V, the monitoring mode adopts a multi-reaction detection mode MRM, and the concentration of parent ions: 1257.7, daughter ion: 764.4, 650.4.
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| US8106172B2 (en) * | 2003-06-10 | 2012-01-31 | Biomerieux, B.V. | Nucleic acid sequences that can be used as primers and probes in the amplification and detection of SARS coronavirus |
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| CN109444287A (en) * | 2018-12-20 | 2019-03-08 | 杭州同创医学检验实验室有限公司 | A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
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