Method for rapidly identifying avian leukosis virus

Abstract

The invention relates to the field of avian virus detection, and discloses a method for rapidly identifying avian leukosis virus, which comprises the following steps: S1, sample collection and pretreatment: firstly collecting a poultry sample, S2, loading a micro-fluidic chip, and then injecting the pretreated sample into the micro-fluidic chip; s3, performing micro-fluidic LAMP amplification on the micro-fluidic chip to obtain enough products for subsequent detection; s4, a CRI SPR specific detection amplification product automatically enters a CRI SPR reaction chamber in the chip; and S5, collecting and analyzing a detection signal, and reading a fluorescence signal on the chip by using a portable fluorescence detector. By fusing the rapid amplification capability of the microfluidic LAMP and the high-specificity detection advantage of CRISPR / Cas12a, the avian leukosis virus can be rapidly, sensitively and specifically recognized, the portable and field application is realized, the target sequence in the amplification product is recognized, the cleavage function is activated, and the detection sensitivity is high. In use, whether a sample reacted by a fluorescence signal contains viruses or not can be quickly identified.

Description

Technical Field

[0001] The invention relates to the field of avian virus detection, in particular to a method for rapidly identifying avian leukosis virus. Background Art

[0002] Avian leukosis virus is a type of virus that can infect birds, especially poultry, and belongs to the family of retroviruses. It is one of the main causes of avian leukosis, tumors and other related diseases. Avian leukosis virus is divided into different subtypes, and the types of diseases and symptoms caused by different subtypes are different.

[0003] Avian leukosis is one of the diseases that seriously harm the poultry farming industry. It is caused by the avian leukosis virus and has the characteristics of wide spread and great harm. Accurate and rapid detection of avian leukosis virus is the key to effectively prevent and control the disease and ensure the healthy development of the poultry farming industry.

[0004] Traditional detection methods usually rely on complex laboratory equipment and cumbersome operating procedures. For example, although the detection technology based on polymerase chain reaction has a certain degree of accuracy, it requires precise temperature cycle control, and the instruments and equipment are expensive and complicated to operate, making it difficult to apply quickly on site. Although the serological detection method is relatively simple to operate, its sensitivity and specificity are often not ideal, and false positive or false negative results are prone to occur. Some emerging detection technologies, such as gene chip technology, although they can detect multiple pathogens at the same time, have problems such as high detection costs and complex data analysis, and cannot effectively prevent and control the spread of avian leukosis virus in a timely manner. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for rapidly identifying avian leukosis virus, which solves the problems that traditional detection methods rely on large laboratory equipment, are not convenient for on-site instant detection, limit their application in on-site environments such as farms, have cumbersome and complicated operation procedures, require professional technicians to operate, increase labor costs and time costs, and are difficult to balance the sensitivity and specificity of detection, which may result in misjudgment, and some emerging detection technologies are expensive.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: A method for rapidly identifying avian leukosis virus comprises the following steps:

[0007] S1, sample collection and pretreatment, first collect poultry samples, then put the samples into a container containing rapid lysis solution and RNA stabilization solution, mix them thoroughly and complete RNA release. RNA release is used to ensure the integrity of nucleic acid and adapt to the role of on-site instant detection;

[0008] S2 microfluidic chip loading, then the pre-treated sample is injected into the microfluidic chip, which has multiple reaction chambers, and then the sample is automatically distributed to each reaction chamber by capillary action;

[0009] S3 microfluidic LAMP amplification obtains enough product for subsequent detection in the microfluidic chip, and uses LAMP technology to amplify the target RNA fragment;

[0010] The S4CR I SPR specific detection amplification product automatically enters the CRI SPR reaction chamber in the chip, and the Cas12a protein binds to the specific crRNA, recognizes the target sequence in the amplification product and activates the cleavage function. The cleavage reaction uses an FQ fluorescent probe containing a fluorescent group and a quenching group;

[0011] S5 detection signal acquisition and analysis uses a portable fluorescence detector to read the fluorescence signal on the chip, supports automatic recording of results and generation of reports, which are used to quickly determine on-site whether the sample has viral effects.

[0012] Preferably, in S1, the poultry samples include blood, feathers and tissue fluid samples, the rapid lysis solution contains detergents, salts, protease inhibitors and nucleic acid protectants, and the RNA stabilization solution includes chemical inhibitors, chelating agents and buffer components, wherein the mixing of the container completes the RNA release time of 2-5 minutes.

[0013] Preferably, in S2, the avian leukosis virus subtypes include subtype A, subtype B, subtype J and subtype K, and a plurality of reaction chambers are provided in the chip, corresponding to specific LAMP primers and related reagents of different virus subtypes, respectively. The related reagents generally include polymerase, deoxyribonucleoside triphosphates, buffer and betaine, and the reaction chambers of the microfluidic chip are used to uniformly and automatically distribute samples by capillary action.

[0014] Preferably, in S3, the constant temperature condition of the microfluidic LAMP amplification is 65° C., the amplification time is 20 minutes, and the RNA fragment includes the LTR region or the env gene fragment.

[0015] Preferably, in S4, in the CRI SPR specific detection, after the Cas12a protein binds to the specific crRNA, it activates the cleavage of the FQ fluorescent probe containing a fluorescent group and a quenching group to produce a fluorescent signal only when the target sequence is present, and the fluorescent signal is used to determine whether the sample is positive or negative.

[0016] Preferably, in S5, the detection time of the fluorescence detector is 30-40 minutes, and the fluorescence signal shows a strong fluorescence signal in the positive reaction chamber and no fluorescence in the negative reaction chamber.

[0017] The present invention provides a method for rapidly identifying avian leukosis virus, which has the following beneficial effects:

[0018] 1. The present invention combines the rapid amplification capability of microfluidic LAMP and the high specificity detection advantages of CRISPR / Cas12a to quickly, highly sensitively and specifically identify avian leukosis virus, realize portable and on-site applications, and simultaneously identify the target sequence in the amplified product and activate the cutting function. During use, it can quickly identify whether the sample contains the virus through the fluorescent signal reaction.

[0019] 2. The method of the present invention does not require complicated laboratory conditions. Combined with the use of rapid lysis solution and RNA stabilization solution, sampling can be done directly in poultry farms or on-site and RNA can be quickly released, shortening the sample processing time to 2-5 minutes. The microfluidic chip realizes automatic sample distribution, eliminating the need for manual operation and improving the detection efficiency.

[0020] 3. The present invention can achieve early warning by rapidly detecting multiple subtypes of avian leukosis virus and outputting high-precision results, helping poultry farms to take isolation and treatment measures in time, reducing virus transmission and economic losses. It can also be used to detect other avian viruses or related pathogens by adjusting the crRNA design of LAMP primers and CRISPR / Cas12a, and has wide applicability and technical expansion potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a method flow chart of a method for rapidly identifying avian leukosis virus. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example:

[0024] Please refer to the attached Figure 1 The embodiment of the present invention provides a method for quickly identifying avian leukosis virus, comprising the following steps:

[0025] S1, sample collection and pretreatment, first collect poultry samples, then put the samples into a container containing rapid lysis solution and RNA stabilization solution, mix them thoroughly and complete RNA release. RNA release is used to ensure the integrity of nucleic acid and adapt to the role of on-site instant detection;

[0026] S2 microfluidic chip loading, then the pre-treated sample is injected into the microfluidic chip, which has multiple reaction chambers, and then the sample is automatically distributed to each reaction chamber by capillary action;

[0027] S3 microfluidic LAMP amplification obtains enough product for subsequent detection in the microfluidic chip, and uses LAMP technology to amplify the target RNA fragment;

[0028] The S4CR I SPR specific detection amplification product automatically enters the CRI SPR reaction chamber in the chip, and the Cas12a protein binds to the specific crRNA, recognizes the target sequence in the amplification product and activates the cleavage function. The cleavage reaction uses an FQ fluorescent probe containing a fluorescent group and a quenching group;

[0029] S5 detection signal acquisition and analysis uses a portable fluorescence detector to read the fluorescence signal on the chip, supports automatic recording of results and generation of reports, which are used to quickly determine on-site whether the sample has viral effects.

[0030] In S1, poultry samples include blood, feathers and tissue fluid samples, the rapid lysis solution contains detergents, salts, protease inhibitors and nucleic acid protectants, and the RNA stabilization solution includes chemical inhibitors, chelating agents and buffer components, wherein the mixing of the container completes the RNA release time of 2-5 minutes.

[0031] Specifically, poultry samples include blood, feathers, tissue fluid and other forms. Rapid lysis solution is composed of detergents, salts, protease inhibitors and nucleic acid protectants. Among them, detergents can quickly destroy the membrane structure of cells and release the components in cells; salts help maintain a suitable ionic environment to ensure the smooth progress of the reaction; protease inhibitors can effectively inhibit the activity of proteases and prevent protein degradation; nucleic acid protectants can protect nucleic acids and maintain the integrity of their structure and function.

[0032] RNA stabilization solution contains chemical inhibitors, chelating agents and buffer components. Chemical inhibitors can specifically inhibit the activity of RNases, thereby avoiding RNA degradation; chelating agents reduce the activity of RNases by binding to metal ions, further ensuring the stability of RNA; buffer components can maintain the pH stability of the solution, providing a good chemical environment for RNA, which helps its long-term preservation.

[0033] In this process, the poultry samples are placed in a container containing the above ingredients for mixing, which greatly improves the efficiency of the test and effectively ensures the quality and integrity of the released RNA, providing a reliable basis for subsequent detection and analysis.

[0034] In S2, avian leukosis virus subtypes include subtype A, subtype B, subtype J and subtype K. The chip is equipped with multiple reaction chambers corresponding to specific LAMP primers and related reagents of different virus subtypes. The related reagents usually include polymerase, deoxyribonucleoside triphosphate, buffer and betaine. The reaction chamber of the microfluidic chip is used to uniformly and automatically distribute samples through capillary action.

[0035] Specifically, polymerase has chain displacement activity and can efficiently catalyze nucleic acid amplification reactions under constant temperature conditions; deoxyribonucleoside triphosphates, as the basic raw materials for nucleic acid synthesis, provide the material basis for the amplification reaction; the buffer can maintain the pH value and ionic strength of the reaction system stable, creating a suitable chemical environment for the smooth progress of the reaction; betaine helps to improve the efficiency and specificity of the reaction and enhance the accuracy of detection.

[0036] The reaction chamber of the microfluidic chip has the function of uniformly and automatically distributing samples through capillary action. This capillary action can automatically and evenly distribute samples to each reaction chamber without external intervention. Its function is to ensure equal distribution of samples in different reaction chambers, reduce errors and inhomogeneities caused by manual operation, and thus improve the accuracy and repeatability of the test results. At the same time, this automatic distribution method also greatly improves the efficiency of the test, making the test process more convenient, fast and reliable.

[0037] In S3, the constant temperature condition of microfluidic LAMP amplification was 65°C, the amplification time was 20 minutes, and the RNA fragments included LTR regions or env gene fragments.

[0038] Specifically, microfluidic LAMP amplification is performed in a microfluidic chip to obtain sufficient products for subsequent accurate detection. This process uses efficient LAMP technology to amplify the target RNA fragment. The constant temperature condition of microfluidic LAMP amplification is set to 65°C. It can ensure that the enzyme activity in the LAMP reaction system is in the optimal state, thereby greatly improving the amplification efficiency and accuracy. This relatively short time not only ensures sufficient amplification effect, allowing the target RNA to be replicated in large quantities and produce sufficient product for subsequent detection, but also effectively improves the speed of the entire detection process, meeting the needs of rapid detection.

[0039] The RNA fragments targeted include LTR regions or env gene fragments. These specific regions are selected because they have key identification and specificity in the genetic information of avian leukosis virus. Amplification and detection of these fragments can accurately identify the presence of the virus, providing a reliable basis for accurately determining whether the sample is infected with avian leukosis virus.

[0040] In S4, in CRISPR-specific detection, after the Cas12a protein binds to the specific crRNA, it activates and cuts the FQ fluorescent probe containing a fluorescent group and a quenching group to produce a fluorescent signal only when the target sequence is present. The fluorescent signal is used to determine whether the sample is positive or negative.

[0041] Specifically, in the key link of CRISPR specific detection, the Cas12a protein is tightly bound to the specific crRNA. This binding is highly specific and accurate, and only in the presence of the target sequence will it activate the cleavage of the FQ fluorescent probe containing the fluorescent group and the quenching group;

[0042] The combination of Cas12a protein and specific crRNA is like a precise key. Only when it encounters a lock with a target sequence that matches it completely, will the subsequent cutting reaction be initiated. However, the FQ fluorescent probe containing a fluorescent group and a quenching group will not emit fluorescence when it is not cut due to the presence of the quenching group. Once the Cas12a protein binds to the specific crRNA and recognizes the target sequence, the cutting action is activated, the fluorescent group and the quenching group are separated, and the fluorescent signal is released;

[0043] The generated fluorescent signal plays a vital role in the detection. It is the key basis for determining whether the sample is positive or negative. If a strong fluorescent signal is detected, it indicates that the target sequence exists in the sample, that is, the sample is positive, which means that it may be infected with avian leukosis virus; conversely, if no fluorescent signal is detected or the fluorescent signal is extremely weak, it means that the sample is negative and is most likely not infected with the virus. This method of judging by fluorescent signals has extremely high sensitivity and accuracy, and can provide reliable results for the detection of avian leukosis virus.

[0044] In S5, the detection time of the fluorescence detector is 30-40 minutes, and the fluorescence signal shows a strong fluorescence signal in the positive reaction chamber and no fluorescence in the negative reaction chamber.

[0045] Specifically, for the test results, the fluorescent signal presents obvious distinguishing characteristics. In the positive reaction chamber, a strong fluorescent signal is displayed. This strong and significant fluorescence indicates that there is a target sequence related to avian leukosis virus in the sample, that is, the sample is judged as positive, suggesting that there may be infection with avian leukosis virus. In the negative reaction chamber, there is no fluorescence at all. This zero fluorescence state clearly indicates that the relevant sequence of the target virus is not detected in the sample, so it is judged as negative, indicating that the sample is most likely not infected with avian leukosis virus. This method of clearly distinguishing positive and negative reactions by the presence and strength of fluorescent signals has extremely high accuracy and reliability, and provides strong support for the rapid and effective detection of avian leukosis virus.

[0046] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapidly identifying avian leukosis virus, characterized in that: The following steps are involved: S1. Sample collection and pretreatment: first collect poultry samples, then place the samples in a container containing a rapid lysis solution and an RNA stabilizing solution, mix them thoroughly and release RNA. RNA release is used to ensure the integrity of nucleic acids and adapt to the role of on-site instant detection; S2, loading the microfluidic chip, then injecting the pre-treated sample into the microfluidic chip, which has multiple reaction chambers, and then automatically distributing the sample to each reaction chamber using capillary action; S3, microfluidic LAMP amplification: sufficient product is obtained on the microfluidic chip for subsequent detection, and the target RNA fragment is amplified using LAMP technology; S4, CRISPR specific detection: The amplified product automatically enters the CRISPR reaction chamber in the chip, and the Cas12a protein binds to the specific crRNA, recognizes the target sequence in the amplified product and activates the cleavage function. The cleavage reaction uses an FQ fluorescent probe containing a fluorescent group and a quenching group; S5. Detection signal acquisition and analysis: Use a portable fluorescence detector to read the fluorescence signal on the chip, support automatic recording of results and generate reports, which are used to quickly determine on-site whether the sample has the effect of the virus.

2. A method for rapidly identifying avian leukosis virus according to claim 1, characterized in that: In S1, the poultry samples include blood, feathers and tissue fluid samples, the rapid lysis solution contains detergents, salts, protease inhibitors and nucleic acid protectants, and the RNA stabilization solution includes chemical inhibitors, chelating agents and buffer components, wherein the mixing of the container completes the RNA release time of 2-5 minutes.

3. A method for rapidly identifying avian leukosis virus according to claim 1, characterized in that: In S2, the avian leukosis virus subtypes include subtype A, subtype B, subtype J and subtype K. A plurality of reaction chambers are provided in the chip, which correspond to specific LAMP primers and related reagents of different virus subtypes. The related reagents generally include polymerase, deoxyribonucleoside triphosphate, buffer and betaine. The reaction chamber of the microfluidic chip is used to uniformly and automatically distribute the sample by capillary action.

4. A method for rapid identification of avian leukosis virus according to claim 1, characterized in that: In S3, the constant temperature condition of the microfluidic LAMP amplification is 65° C., the amplification time is 20 minutes, and the RNA fragment includes the LTR region or the env gene fragment.

5. A method for rapid identification of avian leukosis virus according to claim 1, characterized in that: In S4, in the CRISPR-specific detection, after the Cas12a protein binds to the specific crRNA, it activates and cuts the FQ fluorescent probe containing a fluorescent group and a quenching group to generate a fluorescent signal only when the target sequence is present. The fluorescent signal is used to determine whether the sample is positive or negative.

6. A method for rapid identification of avian leukosis virus according to claim 1, characterized in that: In S5, the detection time of the fluorescence detector is 30-40 minutes, and the fluorescence signal shows a strong fluorescence signal in the positive reaction chamber and no fluorescence in the negative reaction chamber.

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