Critical viral infection identification method and application

Abstract

The application provides a key virus infection identification method and application, and relates to the technical field of virus nucleic acid detection. The method comprises the following steps: S1, collecting samples, and dividing the samples into three parts after pretreatment; S2, extracting nucleic acid from the first sample, directly performing qPCR reaction, detecting CT value as Ct1; adding nucleic acid dye to the second sample for incubation and photolysis, extracting nucleic acid, performing qPCR reaction, and detecting CT value as Ct2; adding nucleic acid dye to the third sample after inactivation at 90 DEG C for incubation and photolysis, extracting nucleic acid, performing qPCR reaction, and detecting CT value as Ct3; S3, analyzing, calculating and judging the infectivity of viruses in the sample according to the detected CT values. The identification method can be used for disinfection effect determination, and the determination result is accurate and reliable, and the problem of inaccurate result determination caused by the sensitivity difference between nucleic acid dye and different viruses can be effectively avoided.

Description

Technical Field

[0001] This invention relates to the field of viral nucleic acid detection technology, specifically to a key method for identifying viral infectivity and its application. Background Technology

[0002] Currently, pig farms and other livestock farms mostly use spray disinfectant to disinfect vehicles and the environment. However, there is a problem that viral nucleic acid can still be detected after multiple disinfections, which makes it impossible to guarantee the disinfection effect and increases the disinfection cost for front-line workers.

[0003] Currently, the primary method for nucleic acid detection of key viruses in pig farms is qPCR (Quantitative Real-time PCR). This method plays a significant role in virus detection due to its high specificity and sensitivity. However, it can only detect the presence of viral nucleic acid sequences in a sample; it cannot provide effective indications regarding the integrity of the viral capsid protein or its infectivity. Therefore, it is still impossible to accurately determine the disinfection effect. Currently, after detecting viral nucleic acid sequences in a sample, cell assays are typically used to determine whether the contained viral nucleic acid sequences are infectious. However, cell assays are time-consuming, inefficient, and difficult to widely apply. In actual production, after detecting viral nucleic acid sequences in a sample, the disinfection method may be changed and re-disinfected, followed by qPCR testing until the viral nucleic acid sequences are no longer detected, resulting in high disinfection and testing costs. To address these issues, our company has developed a key virus infectivity identification method to rapidly and conveniently determine the disinfection effect. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a key method and application for identifying viral infectivity to determine whether the nucleic acid in a sample is infectious, thereby determining whether disinfection is complete. This method is simple to operate, has a short experimental time, is highly efficient, and provides accurate and reliable results.

[0005] The technical solution adopted in this invention is as follows:

[0006] Key methods for identifying viral infectivity include the following steps:

[0007] S1. Collect samples, preprocess them, and divide them into three portions;

[0008] S2. Nucleic acid was extracted from the first sample and qPCR reaction was performed directly. The CT value was Ct1.

[0009] The second sample was incubated with nucleic acid dye, photolyzed, and nucleic acid was extracted. qPCR was performed, and the CT value was measured as Ct2.

[0010] The third sample was inactivated at 90℃, then incubated with nucleic acid dye, photolyzed, and nucleic acid was extracted. qPCR reaction was performed, and the CT value was detected as Ct3.

[0011] S3. Analyze, calculate and interpret the infectivity of the virus in the sample based on the detected CT value.

[0012] Optionally, in step S3, analyzing, calculating, and interpreting the infectivity of the virus in the sample based on the detected CT value specifically includes:

[0013] The proportion of infectious virus in the sample was calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) x 100%.

[0014] Optionally, the nucleic acid dye is PMA, EMA, or PMAxx.

[0015] Optionally, the key virus includes one or more of ASFV, PEDV, PRRSV, CSFV, and PRV.

[0016] Optionally, when the key virus is ASFV, the nucleotide sequences of the primers in the qPCR reaction are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3;

[0017] When the key virus is PEDV, the nucleotide sequences of the primers in the qPCR reaction are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6;

[0018] When the key virus is PRRSV, the nucleotide sequences of the primers in the qPCR reaction are shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the probe is shown in SEQ ID NO.9.

[0019] Optionally, the formula (Ct3-Ct2) / (Ct3-Ct1)×100% is used to calculate the proportion of infectious virus in the sample only when the viral nucleic acid test result of the sample is positive.

[0020] Optionally, when the key virus is PEDV, PMA is used as the nucleic acid dye, and if Ct1 ≤ 34, the sample is judged to be positive.

[0021] When the key virus is PRRSV, PMA is used as the nucleic acid dye, and if Ct1 ≤ 37, the sample is considered positive.

[0022] When the key virus is ASFV, PMA is used as the nucleic acid dye, and if Ct1 ≤ 36, the sample is considered positive.

[0023] Optionally, in step S2, if the CT value cannot be detected after qPCR reaction of the three samples, the CT value is counted as 40.

[0024] Optionally, in step S2, the incubation and photolysis specifically include:

[0025] The third sample was heated at 90°C for 10 minutes to inactivate the virus, and then cooled to room temperature.

[0026] Add nucleic acid dye to the second sample and the inactivated third sample in the dark, and mix well.

[0027] Add an equal volume of ddH2O to the first sample and mix well;

[0028] Three samples were incubated at 37°C in the dark for 13-20 minutes, and then cooled on ice.

[0029] Then place it in a photolysis apparatus and photolyze for 15-20 minutes.

[0030] The application of any of the key viral infectivity identification methods described above can be used to determine the effectiveness of disinfection of farm environments and items.

[0031] The basic principle is that nucleic acid dyes are positively charged molecules that are rejected by intact, negatively charged bacterial cell walls / membranes / capsid proteins. However, they can enter bacteria with damaged cell walls / membranes / capsid proteins. During dark incubation, the dye binds to DNA / RNA in a valence state. After exposure treatment, the dye modifies the DNA / RNA, changing its structure and thus inhibiting subsequent amplification.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] The method of this invention divides the sample into three parts and performs qPCR directly, qPCR after treatment with nucleic acid dye, and qPCR after inactivation with nucleic acid dye. The infectivity of the sample is determined based on changes in the CT value. This method provides accurate and reliable results, effectively avoiding inaccurate results caused by differences in sensitivity between nucleic acid dyes and different viruses. It also effectively eliminates the false positive problem associated with existing V-PCR methods (which directly add nucleic acid dye for qPCR to detect CT values; if no CT value is detected or the CT value exceeds a certain value, it is considered non-infectious; otherwise, it is considered infectious, i.e., positive). This method can be used to determine the effectiveness of disinfection, and it is rapid and effective. When used to determine the effectiveness of disinfection in livestock farms, it can accelerate the resumption of production, reduce disinfection and testing costs, and is highly applicable and easy to promote and apply. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The amplification curves of the PRRSV vaccine sample in Experiment Example 1 after treatment with different nucleic acid dyes are shown.

[0036] Figure 2 The amplification curves of PRDV field samples treated with different nucleic acid dyes in Experiment Example 1 are shown.

[0037] Figure 3 The amplification curves of PMRV vaccine samples with different dilution gradients in Experiment Example 2 after PMA treatment are shown.

[0038] Figure 4 The amplification curves (I) of PRDV field samples with different dilution gradients after PMA treatment in Experiment Example 2 are shown.

[0039] Figure 5 The amplification curves (II) of PRDV field samples with different dilution gradients after PMA treatment are shown in Experiment Example 2.

[0040] Figure 6 The amplification curves (I) of ASFV samples with different dilution gradients after PMA treatment in Experiment Example 2 are shown.

[0041] Figure 7 The amplification curves (II) of ASFV samples with different dilution gradients after PMA treatment in Experiment Example 2 are shown.

[0042] Figure 8 The amplification curves (III) of ASFV samples with different dilution gradients after PMA treatment in Experiment Example 2. Detailed Implementation

[0043] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0044] Example 1

[0045] This invention discloses a key method for identifying viral infectivity. This method is applicable to identifying whether viral nucleic acid exists in a sample and whether the viral nucleic acid is live and infectious.

[0046] The method includes the following steps:

[0047] Step S1: Collect samples, preprocess the samples, and then divide them into three portions for later use.

[0048] The sample must be in liquid form. If the sample is a swab, wash the swab with enzyme-free PBS or ddH2O and collect the eluent. Take a certain volume of liquid sample or eluent, centrifuge it, collect the supernatant, and divide the supernatant into three portions for later use. If the sample has insufficient supernatant, make up the amount with enzyme-free PBS or ddH2O.

[0049] Step S2: Extract nucleic acid from the first sample using a commercially available kit, and directly perform qPCR amplification reaction on the extracted nucleic acid sample to detect the CT value, which is recorded as Ct1.

[0050] Nucleic acid dye was added to the second sample, incubated, and photolyzed. Then, nucleic acid was extracted using a commercially available kit. The extracted nucleic acid sample was subjected to qPCR amplification, and the CT value was detected. This CT value was recorded as Ct2.

[0051] The third sample was heated at 90℃ for 10 min to inactivate the virus, then nucleic acid dye was added, and the sample was incubated and photolyzed. Nucleic acid was then extracted using a commercially available kit. The extracted nucleic acid sample was subjected to qPCR amplification and the CT value was detected. This CT value was recorded as Ct3.

[0052] Specifically, during the incubation and photolysis process, firstly, 190 μL of each of the three samples were placed into three 1.5 mL enzyme-free centrifuge tubes. Secondly, the third sample was heated at 90 °C for 10 min to inactivate the virus, and then cooled to room temperature. Next, 10 μL of nucleic acid dye was added to each of the second and inactivated third samples in the dark, and 10 μL of ddH2O of equal volume to the nucleic acid dye was added to the first sample, and the mixture was stirred well. Then, the three samples were incubated at 37 °C in the dark for 13-20 min, and immediately placed on ice water to cool after incubation. Finally, the three samples were placed in a photolysis apparatus and photolyzed for 15-20 min. After photolysis, no further light protection was required.

[0053] It should be noted that when the key virus is a DNA virus, the incubation time in the dark is 15-20 minutes, and the photolysis time is 15-20 minutes; when the key virus is an RNA virus, the incubation time in the dark is 13-15 minutes, and the photolysis time is 18-20 minutes under a 100W blue light lamp. During incubation and photolysis, the mixture should be shaken and mixed once every 4-6 minutes.

[0054] Step S3: Analyze, calculate and interpret the infectivity of the virus in the sample based on the CT values ​​(Ct1, Ct2, Ct3) detected in step S2.

[0055] Specifically, the proportion of infectious virus in the sample is calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100%, thereby determining whether the sample contains infectious virus (active virus) and how much infectious virus it contains.

[0056] When calculating the proportion of infectious virus in a sample using the formula (Ct3-Ct2) / (Ct3-Ct1) x 100%, this formula should only be used when the viral nucleic acid test result of the sample is positive, indicating that the nucleic acid contained in the sample can be amplified and detected. Otherwise, it has no diagnostic significance.

[0057] Furthermore, to ensure the accuracy of this method, the CT value (i.e., Ct1) obtained after qPCR amplification of the first sample in step S2 is used for evaluation. When the Ct1 value is less than or equal to a specific threshold, the viral nucleic acid test result of the sample is considered positive. When the Ct1 value of the sample is less than or equal to the specific threshold, the infectivity of the virus can be determined using the corresponding nucleic acid dye; when the Ct1 value of the sample is greater than the specific threshold, the corresponding CT value cannot be amplified and detected after adding the nucleic acid dye, or the CT value does not change (the change is less than 0.5).

[0058] The nucleic acid dye used in this method can be ethidium monoazide (EMA), propidium monoazide (PMA), or PMA derivatives such as PMAxx; PMA is preferred. That is, PMA, PMAxx, and EMA can all bind to nucleic acids in the sample, increasing the CT value of the sample, but PMA has a better binding rate, resulting in a larger ΔCT compared to the original CT value of the sample.

[0059] This method can be applied to detect any one or more of the following viruses: African swine fever virus (ASFV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and pseudorabies virus (PRV). In other words, the key virus identified by this method can be one or more of ASFV, PEDV, PRRSV, CSFV, and PRV.

[0060] When the key virus being identified is ASFV, the nucleotide sequence of the upstream primer in the qPCR reaction is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the specific probe is shown in SEQ ID NO.3. The 5' end and 3' end of the probe are respectively bound to the fluorescent generating group FAM and the fluorescent quenching group TAMRA.

[0061] When the key virus being identified is PEDV, the nucleotide sequence of the upstream primer in the qPCR reaction is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the specific probe is shown in SEQ ID NO.6. The 5' end and 3' end of the probe are respectively bound to the fluorescent generating group FAM and the fluorescent quenching group DBQ1.

[0062] When the key virus being identified is PRRSV, the nucleotide sequence of the upstream primer in the qPCR reaction is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, and the nucleotide sequence of the specific probe is shown in SEQ ID NO.9. The 5' end and 3' end of the probe are respectively bound to the fluorescent generating group FAM and the fluorescent quenching group DBQ.

[0063] When the key virus to be identified is PEDV, PMA is used as the nucleic acid dye. A Ct1 value ≤ 34 indicates a positive viral nucleic acid test result for the sample. The proportion of infectious virus in the sample can be calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100%. In other words, when using PMA as a nucleic acid dye to identify the infectivity of PEDV in a sample, the Ct1 threshold is 34.

[0064] When the key virus to be identified is PRRSV, PMA is used as the nucleic acid dye. A Ct1 value ≤ 37 indicates a positive viral nucleic acid test result for the sample. The proportion of infectious virus in the sample can be calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100%. In other words, when using PMA as a nucleic acid dye to identify the infectivity of PRRSV in a sample, the Ct1 threshold is 37.

[0065] When the key virus to be identified is ASFV, PMA is used as the nucleic acid dye. A Ct1 value ≤ 36 indicates a positive viral nucleic acid test result for the sample. The proportion of infectious virus in the sample can be calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100%. In other words, when using PMA as a nucleic acid dye to identify the infectivity of ASFV in a sample, the Ct1 threshold is 36.

[0066] Furthermore, in step S2, if the three samples do not amplify after qPCR reaction and their CT values ​​cannot be detected, the CT value is counted as 40 and then used in the formula (Ct3-Ct2) / (Ct3-Ct1)×100% for calculation.

[0067] Results analysis and interpretation:

[0068] When performing ASFV testing, if the Ct1 value of the first sample is less than or equal to 36, it indicates that the sample is positive for ASFV nucleic acid, and the result can be interpreted using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100%. If the Ct1 value is greater than 36, it indicates that the sample contains no ASFV nucleic acid or has a low content, and it is directly interpreted as having no infectious ASFV virus. When Ct2 and Ct3 cannot be detected, they are counted as 40. The result calculated by the formula is less than or equal to 0, which indicates that there is no infectious ASFV virus. The result greater than 0 indicates that the sample contains infectious ASFV virus. The larger the result, the higher the proportion of infectious ASFV virus, and vice versa.

[0069] When performing PEDV testing, if the Ct1 value of the first sample is less than or equal to 34, it indicates that the sample is positive for PEDV nucleic acid. The formula (Ct3-Ct2) / (Ct3-Ct1) × 100% can be used for interpretation. When Ct2 and Ct3 cannot be detected, it is counted as 40. The result calculated by the formula is less than or equal to 0, which indicates that there is no infectious PEDV virus. The result is greater than 0, which indicates that the sample contains infectious PEDV virus. The larger the result, the higher the proportion of infectious PEDV virus, and vice versa.

[0070] When performing PRRSV testing, if the Ct1 value of the first sample is less than or equal to 37, it indicates that the sample is positive for PRRSV nucleic acid, and the result can be interpreted using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100%. If the Ct1 value is greater than 37, it indicates that the sample contains no PRRSV nucleic acid or has a low content, and it is directly interpreted as having no infectious ASFV virus. When Ct2 and Ct3 cannot be detected, they are counted as 40. The result calculated by the formula is less than or equal to 0, which indicates that there is no infectious PRRSV virus. The result greater than 0 indicates that the sample contains infectious PRRSV virus. The larger the result, the higher the proportion of infectious PRRSV virus, and vice versa.

[0071] In summary, this method can be used to determine the infectivity of DNA viruses (such as ASFV) in samples, as well as the infectivity of RNA viruses (such as PEDV and PRRSV) within a certain CT value (Ct1 value) range. The results are accurate and reliable, effectively avoiding inaccurate results caused by differences in sensitivity between nucleic acid dyes and different viruses. Furthermore, it effectively eliminates the false-positive problem inherent in existing V-PCR methods (which directly add nucleic acid dyes for qPCR detection of CT values; if no CT value is detected or the CT value is greater than a specific value, it is considered non-infectious; otherwise, it is considered infectious, i.e., positive).

[0072] Example 2

[0073] This embodiment discloses the application of a key viral infectivity identification method, which can be applied to verify the disinfection effect of farm environment and items, and to determine the viral infectivity of samples, and is especially suitable for verifying the disinfection effect of pig farm environment and items.

[0074] Specifically, samples T1-1 and T1-2 were obtained by swabbing truck wheels after disinfection with disinfectant spray in the pig farm; samples T2-1 and T2-2 were obtained by swabbing pen fences and floors after disinfection with disinfectant spray; samples T3-1 and T3-2 were obtained by swabbing the soles of shoes and boots after disinfection in a disinfection pool; and PEDV positive solution samples were used as control samples T0-1 and T0-2. Samples T1-1, T1-2, T2-1, T2-2, T3-1, and T3-2 were eluted with enzyme-free PBS, and the eluent was collected. The eluent was centrifuged for 2 minutes to collect the supernatant. For samples with less than 600 μL of supernatant, the volume was increased to 600 μL with enzyme-free PBS for later use.

[0075] Take three 190 μL aliquots of each sample solution. Extract nucleic acid from one sample using a commercially available kit, and directly perform qPCR amplification. Detect the CT value, which is recorded as Ct1. Add nucleic acid dye PMA to another sample, incubate, and photolyze. Then extract nucleic acid using a commercially available kit. Perform qPCR amplification on the extracted nucleic acid sample (using PEDV primers and a specific probe), and detect the CT value, which is recorded as Ct2. Inactivate the remaining sample by heating at 90°C for 10 min, then add nucleic acid dye PMA, incubate, and photolyze. Extract nucleic acid using a commercially available kit, and perform qPCR amplification on the extracted nucleic acid sample (using PEDV primers and a specific probe). Detect the CT value, which is recorded as Ct3.

[0076] The results of PEDV infectivity detection for each sample after treatment with the nucleic acid dye PMA are shown in Table 1. Table 1 shows that PEDV nucleic acid sequences were detected in all samples. Specifically, the truck wheels (T1-1 and T1-2) after disinfection with sprayed disinfectant and the soles of shoes after disinfection in a disinfection pool (T3-1 and T3-2) carried a certain proportion of infectious PEDV, indicating incomplete disinfection. The animal enclosure fences and ground (T2-1 and T2-2) after disinfection with sprayed disinfectant did not contain infectious PEDV, indicating complete disinfection. The PEDV-positive solution samples (T0-1 and T0-2) contained a high proportion of infectious ASFV (85.47% and 90.48%, respectively).

[0077] Table 1 shows the results of ASFV infectivity detection for each sample after treatment with the nucleic acid dye PMA.

[0078]

[0079] Note: *The proportion of infectious viruses is calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) x 100%.

[0080] Experimental Example 1

[0081] like Figure 1 Table 2 shows the qPCR results of PRRSV vaccine samples after treatment with different nucleic acid dyes.

[0082] Table 2. CT value detection results of PRRSV vaccine samples after treatment with different nucleic acid dyes.

[0083] Nucleic acid dyes Handling method CT value Original sample - without nucleic acid dye Uninactivated 20.24 PMA Uninactivated 20.27 PMA Inactivation 25.30 PMAxx Uninactivated 19.71 PMAxx Inactivation 23.81 EMA Uninactivated 20.75 EMA Inactivation 25.36

[0084] like Figure 2 Table 3 shows the qPCR results of PEDV field samples after treatment with different nucleic acid dyes.

[0085] Table 3. CT value detection results of PEDV field samples after treatment with different nucleic acid dyes.

[0086] Nucleic acid dyes Handling method CT value Original sample - without nucleic acid dye Uninactivated 25.40 PMA Uninactivated 24.31 PMA Inactivation 33.48 PMAxx Uninactivated 24.93 PMAxx Inactivation 30.12 EMA Uninactivated 25.04 EMA Inactivation 29.91

[0087] In summary, as Figure 1-2 As shown in Tables 2-3, after inactivation, both PRRSV and PEDV field samples showed an increase in CT values ​​after treatment with nucleic acid dyes PMA, PMAxx, and EMA, with the difference in CT values ​​being greater after treatment with nucleic acid dye PMA.

[0088] Experimental Example 2

[0089] like Figure 3Table 4 shows the qPCR results of PRRSV vaccine samples with different dilution gradients after treatment with the nucleic acid dye PMA.

[0090] Table 4. CT value detection results of PRRSV vaccine samples with different dilution gradients after treatment with nucleic acid dye PMA.

[0091]

[0092] In summary, by Figure 3 As shown in Table 4, for serially diluted PRRSV samples, after the original sample CT value (i.e., Ct1 value) reaches 37, the sample CT values ​​(Ct2 and Ct3) no longer change or the change in CT values ​​(Ct2 and Ct3) is less than 0.5 after PMA treatment.

[0093] Furthermore, cell infection assays have shown that PRRSV samples are no longer infectious when the Ct1 value reaches 37. In other words, when the Ct1 value is greater than or equal to 37, the PRRSV infectivity of the sample can be directly determined to be negative.

[0094] Experimental Example 3

[0095] like Figure 4 , Figure 5 Table 5 shows the qPCR results of PEDV field samples with different dilution gradients after treatment with the nucleic acid dye PMA.

[0096] Table 5. CT value detection results of PEDV field samples with different dilution gradients after treatment with nucleic acid dye PMA.

[0097]

[0098] Depend on Figure 4 , Figure 5 As shown in Table 5, after the original CT value (i.e., Ct1 value) of the PEDV graded dilution sample reaches 34, the CT value (Ct2 and Ct3) of the sample cannot be detected after PMA treatment.

[0099] Experiment Example 4

[0100] like Figure 6-8 Table 6 shows the qPCR results of ASFV samples with different dilution gradients after treatment with the nucleic acid dye PMA.

[0101] Table 6. CT value detection results of ASFV samples with different dilution gradients after treatment with nucleic acid dye PMA

[0102]

[0103] Depend on Figure 6-8As shown in Table 6, after the original sample CT value (i.e., Ct1 value) reaches 36, the CT value (Ct2 and Ct3) of the ASFV gradient diluted sample cannot be detected after PMA treatment.

[0104] In the foregoing, only certain exemplary embodiments have been briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of this invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

Claims

1. A key method for identifying viral infectivity, characterized in that, The method described is used to determine the disinfection effect on the environment and items in a breeding farm, and includes the following steps: S1. Collect samples, preprocess them, and divide them into three portions; S2. Nucleic acid was extracted from the first sample and qPCR reaction was performed directly. The CT value was Ct1. The second sample was incubated with nucleic acid dye, photolyzed, and nucleic acid was extracted. qPCR was performed, and the CT value was measured as Ct2. The third sample was inactivated at 90℃, then incubated with nucleic acid dye, photolyzed, and nucleic acid was extracted. qPCR reaction was performed, and the CT value was detected as Ct3. The nucleic acid dye is PMA, EMA, or PMAxx; S3. Analyze, calculate and interpret the infectivity of the virus in the sample based on the detected CT value; The proportion of infectious virus in the sample was calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) x 100%. The proportion of infectious virus in the sample is calculated using the formula (Ct3-Ct2) / (Ct3-Ct1) × 100% only when the viral nucleic acid test result of the sample is positive. When the Ct1 value of a sample is less than or equal to the threshold, the viral nucleic acid test result of the sample is determined to be positive. When Ct2 and Ct3 cannot be detected, Ct2 and Ct3 are counted as 40.

2. The key viral infectivity identification method according to claim 1, characterized in that, The key viruses include one or more of ASFV, PEDV, PRRSV, CSFV, and PRV.

3. The key viral infectivity identification method according to claim 2, characterized in that: When the key virus is ASFV, the nucleotide sequences of the primers in the qPCR reaction are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3; When the key virus is PEDV, the nucleotide sequences of the primers in the qPCR reaction are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6; When the key virus is PRRSV, the nucleotide sequences of the primers in the qPCR reaction are shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the probe is shown in SEQ ID NO.

9.

4. The key viral infectivity identification method according to claim 3, characterized in that: When the key virus is PEDV, PMA is used as the nucleic acid dye, and if Ct1 ≤ 34, the sample is judged to be positive. When the key virus is PRRSV, PMA is used as the nucleic acid dye, and if Ct1 ≤ 37, the sample is considered positive. When the key virus is ASFV, PMA is used as the nucleic acid dye, and if Ct1 ≤ 36, the sample is considered positive.

5. The key viral infectivity identification method according to claim 1, characterized in that, In step S2, incubation and photolysis specifically include: The third sample was heated at 90°C for 10 minutes to inactivate the virus, and then cooled to room temperature. Add nucleic acid dye to the second sample and the inactivated third sample in the dark, and mix well. Add an equal volume of ddH2O to the first sample and mix well; Three samples were incubated at 37°C in the dark for 13-20 minutes, and then cooled on ice. Then place it in a photolysis apparatus and photolyze for 15-20 minutes.

6. The application of the key viral infectivity identification method as described in any one of claims 1 to 5, characterized in that, Used to determine the effectiveness of disinfection of farm environment and items.

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