CN113073146A - Application of BAX full-automatic pathogenic microorganism rapid detection system in African swine fever virus detection - Google Patents
Application of BAX full-automatic pathogenic microorganism rapid detection system in African swine fever virus detection Download PDFInfo
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- CN113073146A CN113073146A CN202110341535.5A CN202110341535A CN113073146A CN 113073146 A CN113073146 A CN 113073146A CN 202110341535 A CN202110341535 A CN 202110341535A CN 113073146 A CN113073146 A CN 113073146A
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- 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
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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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses an application of a BAX full-automatic pathogenic microorganism rapid detection system in African swine fever virus detection, which comprises the following steps: firstly, adding a sample treatment solution into an acquired sample, grinding the sample until the sample is broken, centrifuging the sample, and taking supernatant; secondly, adding DNA extraction liquid into the supernatant, placing the supernatant in a thermal cracking instrument, carrying out constant temperature treatment for 5-6 min under the thermal cracking condition of 97-103 ℃, and then carrying out heat preservation for more than 5min at 2-8 ℃; centrifuging and taking supernatant; thirdly, adding the fluorescent PCR reaction solution into a PCR reaction tube, then adding the supernatant obtained after thermal cracking into the PCR reaction tube, and centrifuging to obtain a sample to be detected; and finally, placing the PCR reaction tube containing the sample to be detected in a reaction chamber of the BAX for on-machine detection. The invention solves the problems of improving the detection capability of a laboratory without increasing experimental equipment and meeting the detection requirement on the African swine fever virus.
Description
Technical Field
The invention belongs to the technical field of African swine fever virus detection, and particularly relates to an application of a BAX full-automatic rapid detection system for pathogenic microorganisms in African swine fever virus detection.
Background
African Swine Fever (ASF) is a virulent infectious disease caused by infecting domestic pigs and wild pigs with African Swine Fever Virus (ASFV), and acute clinical manifestations include high fever, depression, anorexia, cyanosis of skin, bleeding of organs, and the morbidity and mortality can reach 100%. The world animal health Organization (OIE) classifies the animal epidemic disease as a legal report animal epidemic disease, and China classifies the animal epidemic disease as a type of animal epidemic disease and is one of the foreign animal epidemic diseases which are mainly prevented in China. ASF is prevalent in africa, the island of sardine italy, the caucasian area, and in russia and parts of the eastern european countries in the south of sahara, causing significant economic losses to the swine industry in endemic countries and severely impacting the international trade in animal products. In 8 months in 2018, ASF is introduced into China for the first time and then rapidly spread in a large range, which poses a significant threat to the pig industry in China and has extremely severe prevention and control situation. With the global development of economy, the ASF is in a global epidemic situation, and the risk of continuously transferring the ASF into China is extremely high. In view of the fact that the current commercial ASF vaccine is unavailable, the development of an early diagnosis technology capable of realizing rapid detection is urgently needed to realize early discovery and early control of epidemic situation.
The African swine fever virus belongs to the iridovirus of iridoviridae, is a large DNA virus, and the genome of the virus is double-stranded DNA with the size of 170-190 kb. The diameter of the virus particle is 175-225 ms, and the virus particle is 20-face-body symmetrical and provided with a capsule membrane. In pigs, african swine fever virus replicates in several types of cytoplasm, especially in reticuloendothelial cells and mononuclear macrophages. The virus can be propagated in the blunt-pored bees.
At present, a PCR instrument is adopted to detect the African swine fever virus, so that the PCR instrument needs to be purchased separately in the detection process, the detection capability of a laboratory is improved under the condition that experimental equipment is not added, the detection requirement on the African swine fever virus is met, and the problem which needs to be solved urgently is solved.
Disclosure of Invention
In order to solve the problems that the detection capability of a laboratory is improved under the condition that experimental equipment is not added, and the detection requirement on the African swine fever virus is met, the invention provides an application of a BAX full-automatic rapid detection system for pathogenic microorganisms in the detection of the African swine fever virus.
The invention provides an application of a BAX full-automatic pathogenic microorganism rapid detection system in African swine fever virus detection.
Further, the application comprises the following steps:
s1, adding the sample treatment liquid in the African swine fever rapid extraction-free fluorescent PCR detection kit into the collected sample, grinding the sample treatment liquid until the sample treatment liquid is broken, centrifuging the ground sample, and taking supernate;
s2, adding the DNA extract of the African swine fever rapid hands-free fluorescence PCR detection kit into the supernatant obtained in the S1, and placing the mixture into a thermal cracking instrument, wherein the thermal cracking conditions are as follows: after the constant temperature treatment is carried out for 5min to 6min at the temperature of 97 ℃ to 103 ℃, the temperature is preserved for more than 5min at the temperature of 2 ℃ to 8 ℃; subsequently, centrifuging and taking supernatant;
s3, adding the fluorescent PCR reaction solution of the rapid extraction-free fluorescent PCR detection kit for African swine fever into a PCR reaction tube, then adding the supernatant obtained in the step S2 into the PCR reaction tube, and centrifuging to obtain a sample to be detected;
s4, placing the PCR reaction tube containing the sample to be detected in a BAX reaction chamber for on-machine detection.
Further, in the above S1, the centrifugation speed is 9000rpm to 11000rpm, and the centrifugation time is 55S to 65S.
Further, in the step S2, the volume ratio of the DNA extraction solution to the supernatant obtained in the step S1 is 1: 0.8-1.2.
Further, in the S2, the thermal cracking conditions are: after constant temperature treatment at 100 ℃ for 5min, and then heat preservation at 2-8 ℃ for more than 5 min.
Further, in the above S2, the centrifugation speed is 9000rpm to 11000rpm, and the centrifugation time is 55S to 65S.
Further, in S3, the volume ratio of the fluorescent PCR reaction solution to the supernatant obtained in S2 is: 2.5-3.5: 1.
Further, in the step S3, the volume ratio of the fluorescent PCR reaction solution to the supernatant obtained in the step S2 was 3: 1.
Further, in the above S3, the centrifugation speed is 9000rpm to 11000rpm, and the centrifugation time is 8S to 12S.
Compared with the prior art, the invention adopting the scheme has the beneficial effects that:
the steps of the invention are used for processing the pork sample to obtain a sample to be detected, the sample to be detected is placed in a reaction chamber corresponding to BAX, an African swine fever virus detection program is started, detection parameters are set, when the sample to be detected is detected, the detection of positive control and negative control is carried out simultaneously, the laboratory does not detect the African swine fever virus positive sample, the spectrogram of the sample to be detected is often overlapped with the negative control group (line B), so the BAX full-automatic pathogenic microorganism rapid detection system is verified to be applied to the detection of the African swine fever virus, the BAX full-automatic pathogenic microorganism rapid detection system can be directly adopted to detect the African swine fever in the premise that the laboratory only has the BAX full-automatic pathogenic microorganism rapid detection system without a PCR instrument, the detection capability of the laboratory is improved without increasing experimental equipment, and meets the detection requirement of the African swine fever virus.
Drawings
A in figure 1 is a detection spectrogram of a positive control group; in FIG. 1, B is the detection spectrum of the negative control group and the sample to be detected.
Detailed Description
In order that the above objects, features, advantages, etc. of the present invention can be more clearly understood, the present invention will be described in further detail with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
The invention provides an application of a BAX full-automatic pathogenic microorganism rapid detection system in African swine fever virus detection, which specifically comprises the following steps:
s1, adding the sample treatment liquid in the African swine fever rapid extraction-free fluorescent PCR detection kit into the collected sample, grinding the sample treatment liquid until the sample treatment liquid is broken, centrifuging the ground sample, and taking supernate;
wherein the centrifugal speed is 9000 rpm-11000 rpm, and the centrifugal time is 55 s-65 s;
s2, adding the DNA extract of the African swine fever rapid hands-free fluorescence PCR detection kit into the supernatant obtained in the S1, and placing the mixture into a thermal cracking instrument, wherein the thermal cracking conditions are as follows: after the constant temperature treatment is carried out for 5min to 6min at the temperature of 97 ℃ to 103 ℃, the temperature is preserved for more than 5min at the temperature of 2 ℃ to 8 ℃; subsequently, centrifuging and taking supernatant;
wherein the volume ratio of the DNA extraction liquid to the supernatant obtained from S1 is 1: 0.8-1.2;
preferably, the thermal cracking conditions are: after constant temperature treatment at 100 ℃ for 5min, preserving heat at 2-8 ℃ for more than 5 min;
the centrifugal speed is 9000 rpm-11000 rpm, and the centrifugal time is 55 s-65 s;
s3, adding the fluorescent PCR reaction solution of the rapid extraction-free fluorescent PCR detection kit for African swine fever into a PCR reaction tube, then adding the supernatant obtained in the step S2 into the PCR reaction tube, and centrifuging to obtain a sample to be detected;
wherein the volume ratio of the fluorescent PCR reaction solution to the supernatant obtained in S2 is as follows: 2.5-3.5: 1, preferably 3: 1;
the centrifugal speed is 9000 rpm-11000 rpm, and the centrifugal time is 12 s-8 s;
s4, placing the PCR reaction tube containing the sample to be detected in a reaction chamber corresponding to the BAX for on-machine detection.
The following description specifically describes the specific process of using a BAX full-automatic pathogenic microorganism rapid detection system to detect african swine fever virus, with reference to specific examples.
The African swine fever rapid extraction-free fluorescent PCR detection kit used in the application can be obtained through purchase, for example, the African swine fever rapid extraction-free fluorescent PCR detection kit purchased from Guangzhou Rizhen Biotech Limited company, and also purchased from Shenzhen Zhen Rizhen Biotech Limited company. Each African swine fever rapid extraction-free fluorescent PCR detection kit comprises a sample treatment fluid, a DNA extraction fluid and a fluorescent PCR reaction fluid.
Example 1
In order to achieve the above object, this embodiment provides an application of a BAX full-automatic rapid detection system for pathogenic microorganisms in detection of african swine fever virus, including the following steps:
s1, cutting the collected sample into mung bean size, adding 150uL sample treatment liquid in the African swine fever rapid non-extraction fluorescent PCR detection kit, grinding to break, centrifuging at 9000rpm for 65S, and taking supernatant;
s2, taking 40uL of supernatant obtained from S1, adding 50uL of DNA extract of the African swine fever rapid non-extraction fluorescent PCR detection kit (wherein the volume ratio of the DNA extract to the supernatant obtained from S1 is 1:0.8), placing in a thermal cracking instrument, and the thermal cracking conditions are as follows: after constant temperature treatment at 97 ℃ for 6min, preserving heat at 2 ℃ for more than 8 min; subsequently, centrifugation was carried out at 9000rpm for 65s, and the supernatant was taken;
s3, adding the fluorescent PCR reaction solution of the 12.5uL African swine fever rapid non-extraction fluorescent PCR detection kit into a PCR reaction tube, then adding the 5uL supernatant obtained in the step S2 into the PCR reaction tube (wherein the volume ratio of the fluorescent PCR reaction solution to the supernatant in the step S2 is 2.5:1), and centrifuging at 9000rpm for 12S to obtain a sample to be detected;
s4, placing the PCR reaction tube containing the sample to be detected in a BAX reaction chamber for on-machine detection.
Meanwhile, a positive control group and a negative control group are also placed in a reaction chamber of the BAX, a program ABI 7500FAST for African swine fever virus detection is set, program operation detection parameters (such as PCR reaction conditions: pre-denaturation, 1 cycle, 95 ℃/3 min; PCR amplification, 40 cycles, 95 ℃/5sec, 60 ℃/30 sec; fluorescence signal is collected when the temperature is 60 ℃ for extension, a report group is set as FAM, and a quenching group is set as NONE), and finally, the African swine fever virus detection program is started to operate, and detection curves of a sample to be detected, the positive control group and the negative control group appear on a computer display screen, for example, as shown in figure 1.
Example 2
In order to achieve the above object, this embodiment provides an application of a BAX full-automatic rapid detection system for pathogenic microorganisms in detection of african swine fever virus, including the following steps:
s1, cutting the collected sample into mung bean size, adding 150uL sample treatment liquid in the African swine fever rapid non-extraction fluorescent PCR detection kit, grinding the sample treatment liquid to be broken, centrifuging the sample treatment liquid at 10000rpm for 60S, and taking supernatant;
s2, taking 50uL of supernatant obtained from S1, adding DNA extract of the 50uL African swine fever rapid extraction-free fluorescent PCR detection kit (wherein the volume ratio of the DNA extract to the supernatant obtained from S1 is 1:1), placing the mixture in a thermal cracking instrument, and the thermal cracking conditions are as follows: performing constant temperature treatment at 100 deg.C for 5min, and keeping the temperature at 5 deg.C for more than 5 min; subsequently, centrifugation is carried out for 60s at 10000rpm, and supernatant is taken;
s3, adding a fluorescent PCR reaction solution of a 15uL African swine fever rapid extraction-free fluorescent PCR detection kit into a PCR reaction tube, then adding 5uL supernatant obtained in S2 into the PCR reaction tube (wherein the volume ratio of the fluorescent PCR reaction solution to the supernatant in S2 is 3:1), and centrifuging at 10000rpm for 10S to obtain a sample to be detected;
s4, placing the PCR reaction tube containing the sample to be detected in a reaction chamber corresponding to the BAX for on-machine detection.
Meanwhile, a positive control group and a negative control group are also placed in a reaction chamber of the BAX, a program ABI 7500FAST for African swine fever virus detection is set, program operation detection parameters (such as PCR reaction conditions: pre-denaturation, 1 cycle, 95 ℃/3 min; PCR amplification, 40 cycles, 95 ℃/5sec, 60 ℃/30 sec; fluorescence signal is collected when the temperature is 60 ℃ for extension, a report group is set as FAM, and a quenching group is set as NONE), and finally, the African swine fever virus detection program is started to operate, and detection curves of a sample to be detected, the positive control group and the negative control group appear on a computer display screen, for example, as shown in figure 1.
Example 3
In order to achieve the above object, this embodiment provides an application of a BAX full-automatic rapid detection system for pathogenic microorganisms in detection of african swine fever virus, including the following steps:
s1, cutting the collected sample into mung bean size, adding 150uL sample treatment liquid in the African swine fever rapid non-extraction fluorescent PCR detection kit, grinding the sample treatment liquid until the sample treatment liquid is broken, centrifuging the sample treatment liquid for 55S at 11000rpm, and taking supernate;
s2, taking 60uL of supernatant obtained from S1, adding 50uL of DNA extract of the African swine fever rapid non-extraction fluorescent PCR detection kit (wherein the volume ratio of the DNA extract to the supernatant obtained from S1 is 1:1.2), placing the mixture in a thermal cracking instrument, and the thermal cracking conditions are as follows: performing constant temperature treatment at 103 deg.C for 5min, and keeping the temperature at 8 deg.C for more than 5 min; subsequently, centrifugation was carried out at 11000rpm for 55s, and the supernatant was taken;
s3, adding a fluorescent PCR reaction solution of the 17.5uL African swine fever rapid non-extraction fluorescent PCR detection kit into a PCR reaction tube, then adding 5uL supernatant obtained in S2 into the PCR reaction tube (wherein the volume ratio of the fluorescent PCR reaction solution to the supernatant in S2 is 3.5:1), and centrifuging at 11000rpm for 8S to obtain a sample to be detected;
s4, placing the PCR reaction tube containing the sample to be detected in a reaction chamber corresponding to the BAX for on-machine detection.
Meanwhile, a positive control group and a negative control group are also placed in a reaction chamber of the BAX, a program ABI 7500FAST for African swine fever virus detection is set, program operation detection parameters (such as PCR reaction conditions: pre-denaturation, 1 cycle, 95 ℃/3 min; PCR amplification, 40 cycles, 95 ℃/5sec, 60 ℃/30 sec; fluorescence signal is collected when the temperature is 60 ℃ for extension, a report group is set as FAM, and a quenching group is set as NONE), and finally, the African swine fever virus detection program is started to operate, and detection curves of a sample to be detected, the positive control group and the negative control group appear on a computer display screen, for example, as shown in figure 1.
The negative control group represents a sample without African swine fever virus, and the spectra are all a straight line in the effective cycle period during the detection process (as shown in the line B in figure 1); while the positive control group represents the African swine fever virus sample, the spectrum of the sample shows a peak value in the effective cycle period during the detection process (see line A in FIG. 1).
As can be seen from the content shown in fig. 1, the positive control group spectrum (line a) shows a sharp peak within 35 cycles under the same detection conditions and program setting parameters. The spectrum of the negative control (line B) remained a straight line after 35 cycles under the same assay conditions and program set-up parameters.
Therefore, if the same detection conditions and program setting parameters are adopted, and if the sample to be detected is positive, a spectrogram of the sample to be detected inevitably has a steep peak within 35 times of circulation in the detection process. If the sample to be detected is negative, the spectrogram still remains a straight line after 35 cycles in the detection process.
In order to verify the guess, a pork sample is taken, then the pork sample is processed according to the steps of the embodiment 2 to obtain a sample to be detected, meanwhile, a positive control test and a negative control test are carried out, the sample to be detected, the positive control test and the negative control test are placed in a reaction chamber corresponding to the BAX, an African swine fever virus detection program is started, detection parameters are set, the laboratory does not detect the African swine fever virus positive sample, and the fact that the spectrogram of the sample to be detected is often overlapped with a negative control group (line B) is found, so that the BAX full-automatic rapid pathogenic microorganism detection system can be applied to the detection aspect of the African swine fever virus.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
- The application of the BAX full-automatic pathogenic microorganism rapid detection system in the detection of African swine fever virus.
- 2. The application of the BAX full-automatic pathogenic microorganism rapid detection system in the detection of African swine fever virus according to claim 1, which is characterized by comprising the following steps:s1, adding the sample treatment liquid in the African swine fever rapid extraction-free fluorescent PCR detection kit into the collected sample, grinding the sample treatment liquid until the sample treatment liquid is broken, centrifuging the ground sample, and taking supernate;s2, adding the DNA extract of the African swine fever rapid hands-free fluorescence PCR detection kit into the supernatant obtained in the S1, and placing the mixture into a thermal cracking instrument, wherein the thermal cracking conditions are as follows: after the constant temperature treatment is carried out for 5min to 6min at the temperature of 97 ℃ to 103 ℃, the temperature is preserved for more than 5min at the temperature of 2 ℃ to 8 ℃; subsequently, centrifuging and taking supernatant;s3, adding the fluorescent PCR reaction solution of the rapid extraction-free fluorescent PCR detection kit for African swine fever into a PCR reaction tube, then adding the supernatant obtained in the step S2 into the PCR reaction tube, and centrifuging to obtain a sample to be detected;s4, placing the PCR reaction tube containing the sample to be detected in a BAX reaction chamber for on-machine detection.
- 3. The application of the BAX full-automatic rapid detection system for pathogenic microorganisms of claim 1 in the detection of African swine fever virus, wherein in S1, the centrifugation speed is 9000 rpm-11000 rpm, and the centrifugation time is 55S-65S.
- 4. The application of the BAX full-automatic rapid detection system for pathogenic microorganisms in the detection of African swine fever virus according to claim 1, wherein in the S2, the volume ratio of the DNA extraction solution to the supernatant obtained from the S1 is 1: 0.8-1.2.
- 5. The use of the BAX full-automatic rapid detection system for pathogenic microorganisms of claim 1 in the detection of african swine fever virus, wherein in S2, the thermal cracking conditions are as follows: after constant temperature treatment at 100 ℃ for 5min, and then heat preservation at 2-8 ℃ for more than 5 min.
- 6. The application of the BAX full-automatic rapid detection system for pathogenic microorganisms of claim 1 in the detection of African swine fever virus, wherein in S2, the centrifugation speed is 9000 rpm-11000 rpm, and the centrifugation time is 55S-65S.
- 7. The application of the BAX full-automatic pathogenic microorganism rapid detection system in the detection of African swine fever virus according to claim 1, wherein in the S3, the volume ratio of the fluorescent PCR reaction solution to the supernatant obtained from the S2 is as follows: 2.5-3.5: 1.
- 8. The application of the BAX full-automatic pathogenic microorganism rapid detection system in the detection of African swine fever virus according to claim 7, wherein in the S3, the volume ratio of the fluorescent PCR reaction solution to the supernatant obtained from the S2 is 3: 1.
- 9. The application of the BAX full-automatic rapid detection system for pathogenic microorganisms of claim 1 in the detection of African swine fever virus, wherein in S3, the centrifugation speed is 9000 rpm-11000 rpm, and the centrifugation time is 8S-12S.
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