Detection method of porcine cytomegalovirus

Abstract

The invention provides a porcine cytomegalovirus detection method, and belongs to the technical field of biology. According to the invention, a porcine cytomegalovirus screening system is established, and the detection limit of the porcine cytomegalovirus is improved by optimizing quantitative PCR detection parameters. The quantitative PCR shortens the time of the detection technology, and the false positive rate is lower; meanwhile, a PCMV antibody protein immunoblotting detection method is established by matching with a quantitative PCR detection method, and a good detection effect is achieved on individuals with latent infection and non-detoxification. According to the quantitative PCR and western blotting method, the porcine cytomegalovirus of the medical donor pig is detected through a non-invasive sample, the result judgment is more visual, the false positive rate is lower, the accuracy and the sensitivity are high, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a fluorescence quantitative PCR detection method for porcine cytomegalovirus and a method combining the method with protein blotting detection. Background Art

[0002] Porcine cytomegalovirus (PCMV) is an enveloped DNA virus belonging to the family Herpesviridae and the subfamily Betaherpesvirinae. Based on the histopathological characteristics of the disease, PCMV infection is called "inclusion rhinitis". Pigs are the natural host of PCMV and usually carry the virus for life after infection with PCMV, often in a latent state. PCMV infection has not been documented in humans; however, there are concerns about transmission through xenotransplantation. In 2022, the first patient to receive a porcine heart transplant was detected to replicate the virus, and PCMV replication in the patient may have contributed to the patient's eventual death. PCMV is endemic to almost all swine herds in the world, including North America, with seroprevalence rates approaching 100% in many areas. Although newborn piglets can develop fatal systemic disease, adult pigs rarely die. However, co-infection may increase morbidity and mortality. PCMV is transmitted in nasal secretions, ocular secretions, urine, and cervical fluid. Transmission is mainly through direct contact, but congenital transmission also occurs.

[0003] Traditionally, PCMV is diagnosed by histological examination of tissue sections (staining, in situ hybridization, and immunohistochemistry), in which basophilic nuclear inclusions, as well as giant cell disease and nuclear enlargement are usually observed. The polymerase chain reaction (PCR) method for detecting PCMV pathogens and the enzyme-linked immunosorbent assay (ELISA) for PCMV antibody detection have been developed. The main related technologies currently include porcine cytomegalovirus virus colloidal gold test strips, ELISA, LAMP and other detection technologies.

[0004] However, the current detection methods have some shortcomings in practical application. For example, before the first heart transplant patient mentioned above received the transplant, the PCR test result of the donor pig's porcine cytomegalovirus was negative, but after the transplant, the proliferation of porcine cytomegalovirus was detected in the patient's body. This result is caused, on the one hand, by the latent infection characteristics of porcine cytomegalovirus. If it is not in the active period of viral infection, it is difficult to detect the pathogen. On the other hand, during the latent period, the viral load may be lower than the detection limit of the applied PCR method, which has limitations in practical application and cannot guarantee the accuracy of the test results. A more systematic detection method is needed to ensure that the xenotransplant donor pig is PCMV negative.

[0005] The document (DOI:10.3389 / fvets.2020.586045) discloses a PCR detection method for PCMV, but this method requires a total of 50 cycles of PCR reaction, which takes too long, and PCR reactions with too many cycles are prone to produce false positive results.

[0006] In view of the above problems, it is of great significance to develop a method for detecting PCMV that not only takes less time but also has high accuracy and sensitivity. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting porcine cytomegalovirus.

[0008] The present invention provides a fluorescent quantitative PCR detection method for porcine cytomegalovirus, the method comprising the following steps:

[0009] (1) extracting DNA from the sample to be tested as a DNA template;

[0010] (2) performing a PCR reaction: the PCR reaction system includes upstream and downstream primers of porcine cytomegalovirus, a probe and a DNA template;

[0011] (3) performing fluorescence detection;

[0012] When the Ct value of the sample to be tested is ≤35.0, the sample to be tested is judged to be positive; when the Ct value of the sample to be tested is ≥40.0, the sample to be tested is judged to be negative; when the Ct value of the sample to be tested is between 35 and 40, adjust the DNA template concentration and repeat the PCR reaction. When the Ct value of the sample to be tested is <40 after the test, the sample to be tested is judged to be positive, and when the Ct value of the sample to be tested is >40 after the test, the sample to be tested is judged to be negative.

[0013] Furthermore, the method also includes the following steps: setting a blank control, a negative control and a positive control; in the positive control, the PCR reaction system includes upstream and downstream primers, a probe and a plasmid of the porcine cytomegalovirus, and the sequence of the plasmid of the porcine cytomegalovirus is shown in SEQ ID No. 4.

[0014] Furthermore, in step (2), the sequences of the upstream and downstream primers of porcine cytomegalovirus are shown as SEQ ID No.1 and SEQ ID No.2 respectively; and the sequence of the probe of porcine cytomegalovirus is shown as SEQ ID No.3.

[0015] Furthermore, in step (2), the PCR reaction system also includes 1× fluorescent quantitative PCR premix and water.

[0016] Furthermore, the final concentrations of the upstream and downstream primers of porcine cytomegalovirus are 0.1-0.8 μmol / L respectively;

[0017] The final concentration of the porcine cytomegalovirus probe is 0.1-0.8 μmol / L.

[0018] Furthermore, the final concentrations of the upstream and downstream primers of porcine cytomegalovirus are 0.2 μmol / L respectively; and the final concentration of the probe of porcine cytomegalovirus is 0.2 μmol / L.

[0019] Furthermore, in step (2), the PCR reaction procedure is: pre-denaturation at 90-100°C for 20-40s; denaturation at 90-100°C for 2-7s, annealing at 56-64°C for 20-40s; 30-45 cycles.

[0020] Furthermore, in step (2), the PCR reaction procedure is: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 62°C for 30 s; 40 cycles.

[0021] Furthermore, it is characterized in that in step (1), the sample to be tested is pig serum or oral and nasal swab sample.

[0022] The present invention also provides a method for detecting porcine cytomegalovirus, which comprises the following steps:

[0023] (i) Detection is performed according to the above-mentioned fluorescence quantitative PCR detection method for porcine cytomegalovirus;

[0024] (ii) detection using Western blot detection of porcine cytomegalovirus antibodies;

[0025] If the test results of step (i) and step (ii) are both negative, the sample to be tested is judged to be negative.

[0026] Furthermore, step (i) is performed in the first month after the donor pig is born, once a week, for a total of 4 times; step (ii) is performed in the month before the donor pig is used, once every two weeks, for a total of 2 times.

[0027] Further, the Western blot detection method of porcine cytomegalovirus antibodies comprises: pretreatment, electrophoresis and Western blot test;

[0028] The judgment criteria of the protein blotting detection method are: if the negative control has no band signal near 15KDa and the positive control has a band signal near 15KDa, and the test sample has a band signal near 15KDa, the test serum is judged to be positive for porcine cytomegalovirus antibodies, otherwise it is judged to be negative.

[0029] Further, the Western blot test comprises the following steps:

[0030] 1) Protein transfer: transfer the protein to PVDF membrane by wet electroblotting;

[0031] 2) Blocking: Cut the PVDF membrane in 1) into strips and block with buffer at 2-6°C overnight;

[0032] 3) Incubate with primary antibody: dilute the test serum, negative serum and positive serum with blocking solution at 1:200-400, put them into the strips, and incubate at 10-40°C for 1-3 hours;

[0033] 4) Wash the membrane: Place the strips in the buffer solution and wash the membrane for 3 to 7 minutes each time, 2 to 6 times;

[0034] 5) Incubate with secondary antibody: dilute the horseradish peroxidase-labeled antibody in blocking solution at 1:500-1500, place in the strip, and incubate at 10-40°C for 0.5-1.5 hours;

[0035] 6) Repeat the membrane washing and color development;

[0036] The viral protein band signal is 15KDa.

[0037] Further, the Western blot test comprises the following steps:

[0038] 1) Protein transfer: The conditions of wet electroblotting are: 100 V voltage transfer for 1 h;

[0039] 2) Blocking: Cut the PVDF membrane in 1) into strips and block with a buffer solution at 4°C overnight; the buffer solution is a TBST buffer solution containing 5% skimmed milk powder, with a pH of 7.2-7.6;

[0040] 3) Incubate with primary antibody: dilute the test serum, negative serum and positive serum with the blocking solution in 2) at 1:300, put them into the strips, and incubate at 15-35°C for 2 hours;

[0041] 4) Wash the membrane: Place the strips in the buffer solution and wash the membrane for 5 minutes each time, 4 times;

[0042] 5) Incubation with secondary antibody: dilute the antibody labeled with horseradish peroxidase at 1:1000 in blocking buffer, put it into the strip, and incubate at 15-35° C. for 1 hour; the antibody is a rabbit anti-porcine immunoglobulin G polyclonal antibody.

[0043] The present invention has achieved the following beneficial effects:

[0044] The present invention provides a method for detecting porcine cytomegalovirus by fluorescence quantitative PCR. The present invention establishes a porcine cytomegalovirus screening system, improves the detection limit of porcine cytomegalovirus by optimizing the parameters of quantitative PCR detection, and finds that the optimal annealing temperature of the PCR reaction is 62°C and the optimal primer concentration is 0.2 μM. The optimized method for detecting porcine cytomegalovirus by fluorescence quantitative PCR of the present invention not only takes a short time, but also has high accuracy and sensitivity.

[0045] In addition, considering that porcine cytomegalovirus has the characteristic of latent infection, the present invention further provides a method for detecting porcine cytomegalovirus by combining PCMV nucleic acid detection and PCMV antibody detection on the basis of the method for detecting porcine cytomegalovirus by fluorescent quantitative PCR, and the method has a good detection effect for individuals with latent infection and no toxin excretion. The quantitative PCR and protein immunoblotting methods used in the present invention detect porcine cytomegalovirus in medical donor pigs through non-invasive samples, and the result judgment is more intuitive, the false positive rate is lower, the accuracy and sensitivity are higher, and it has good application prospects.

[0046] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0047] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The standard curve is drawn based on the correlation between copy number and CT.

[0049] Figure 2 This is the result of exploring the limiting dilution detection limit (M: protein marker; 1: negative serum 1:300 dilution; 2: positive serum 1:300 dilution; 3: positive serum 1:600 ​​dilution; 4: positive serum 1:1200 dilution; 5: positive serum 1:2400 dilution; 6: positive serum 1:4800 dilution; 7: positive serum 1:9600 dilution; 8: positive serum 1:19200 dilution). DETAILED DESCRIPTION

[0050] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0051] Example 1: Method for detecting porcine cytomegalovirus by fluorescence quantitative PCR

[0052] Use a commercially available DNA extraction kit to extract DNA from pig serum samples, use the probe primers in 1.1, configure the PCR reaction system according to the system configuration table in 1.2, perform the PCR reaction according to the PCR reaction procedure in 1.2, set up positive and negative controls, the positive quality control sample is the plasmid with the target amplification sequence, and the negative quality control sample is the confirmed negative sample. And perform positive and negative determination according to the determination criteria in 1.3.

[0053] 1.1 Primer probe

[0054] The primers used for the probe are as follows:

[0055] Primers:

[0056] PCMV-F:5'-TGGCACTGATACTTGACAAGC-3'(SEQ ID No.1)

[0057] PCMV-R:5'-CTCCCGTGAAGCCGTAAA-3'(SEQ ID No.2)

[0058] Probe:

[0059] 5'FAM-CAGCTTGCCCTCCAAGGTGACGTG-3'BHQ1(SEQ ID No.3)

[0060] 1.2 Reaction system and procedure

[0061] The PCR reaction system was configured according to Table 1, wherein a plasmid with a conserved region sequence of the PCMV genome was obtained by molecular cloning and sent to a sequencing company for sequencing to obtain a specific sequence (SEQ ID No. 4). The theoretical copy number of the plasmid was obtained by a calculation formula, the plasmid was serially diluted, and the serially diluted plasmids were used as templates to perform fluorescence quantitative PCR reactions, and a standard curve was drawn according to the corresponding relationship between the copy number and the CT value.

[0062] PCMV conserved region sequence (SEQ ID No.4)

[0063] .

[0064] Table 1 Fluorescence quantitative PCR reaction system

[0065]

[0066] Reaction procedure: Real-time fluorescence PCR reaction parameters: 94°C pre-denaturation / 30s; 94°C denaturation / 5s, 62°C annealing / 30s, 40 cycles; stored at 4°C after the reaction is completed.

[0067] 1.3 Result determination

[0068] (1) Standard curve

[0069] Table 2 CT values ​​of corresponding copy number plasmids

[0070]

[0071] Depend on Figure 1 It can be seen that the absolute value of the slope of the standard curve 3.331 is very close to the theoretical value 3.32.

[0072] R 2 =0.9982>0.99, indicating that there is a good linear correlation between the logarithm of the copy number with base 10 and the CT value, indicating that the basic system of PCR reaction and the performance of the instrument are good.

[0073] Experimental control settings:

[0074] a) Blank control: no fluorescent signal is detected in the fluorescent channel, and the Ct value should be greater than or equal to 40.0;

[0075] b) Negative control: no fluorescent signal is detected in the fluorescent channel, and the Ct value should be greater than or equal to 40.0;

[0076] c) Positive control: Fluorescence signal is detected in the fluorescence channel, and a typical amplification curve appears, with a Ct value less than 35.0.

[0077] The test results are determined as follows:

[0078] If the Ct value of the sample is less than or equal to 35.0, it is judged as positive; if the Ct value of the sample is greater than or equal to 40.0, it is judged as negative; if the Ct value of the sample is between 35 and 40, the DNA template concentration should be adjusted and the fluorescent quantitative PCR should be repeated. If the Ct value is still less than 40 after re-amplification, it is judged as positive; if the Ct value is greater than 40 after re-amplification, it is judged as negative.

[0079] Example 2: Method for detecting porcine cytomegalovirus by fluorescence quantitative PCR

[0080] The detection method of Example 1 is referred to, except that the extracted sample is replaced from the pig serum sample to the pig oral swab sample.

[0081] Taking into account the latent infection characteristics of porcine cytomegalovirus, the present invention provides a method for detecting porcine cytomegalovirus by combining PCMV nucleic acid detection and PCMV antibody detection in Example 3, based on the method for detecting porcine cytomegalovirus by fluorescent quantitative PCR described in Example 1 and in combination with the PCMV antibody detection method.

[0082] Example 3: Method for detecting porcine cytomegalovirus by combining PCMV nucleic acid detection and PCMV antibody detection

[0083] 1. PCMV nucleic acid detection

[0084] After the donor pigs were born, PCMV nucleic acid testing was performed once a week for 1 month, for a total of 4 times.

[0085] The detection method is the same as in Example 1.

[0086] 2. PCMV antibody detection

[0087] (1) Materials: blood collection tubes containing a coagulant, a group of pigs to be screened, and a PCMV antibody detection kit provided by the present invention.

[0088] (2) Equipment and instruments: centrifuge, micropipette and the following corresponding equipment.

[0089] (3) Method: One month before the donor pig is used, blood is collected from the pig or pig group twice every two weeks using a blood collection tube containing a coagulant. The blood is centrifuged at 3000 RPM for 10 minutes to separate the serum, and the PCMV antibody test is performed using the following PCMV antibody test method:

[0090] Specific methods:

[0091] 1) Protein sample processing

[0092] The sample protein was dissolved in a buffer (50 mM Tris-HCl, 12% glycerol, 4% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue) and denatured at 95°C for 5 minutes before electrophoresis.

[0093] 2) Electrophoresis

[0094] SDS-PAGE was performed using 12.5% ​​polyacrylamide gel.

[0095] 3) Western blot test

[0096] ① Protein transfer: Transfer the protein to the PVDF membrane by wet electroblotting at 100 V for 1 h.

[0097] ② Blocking: Cut into strips and block overnight at 4°C with TBST (Tris 10 mM, NaCl 150 mM, Tween-20 0.05% (V / V), HCl to pH = 7.2-7.6) buffer containing 5% skimmed milk powder.

[0098] ③ Incubate with primary antibody: dilute the test serum, negative serum and positive serum with the above blocking solution at 1:300, place the strips in it and incubate at room temperature for 2 hours.

[0099] ④ Wash the membrane: Place the strips in TBST and wash the membrane on an incubation shaker for 5 minutes each time, for 4 times.

[0100] ⑤ Incubate with secondary antibody: Dilute rabbit anti-swine immunoglobulin G (IgG) polyclonal antibody (Abcam, Cambridge, UK) labeled with horseradish peroxidase at 1:1000 in blocking buffer. Place the strip in it and incubate at room temperature for 1 hour.

[0101] ⑥ Wash the membrane: Place the strips in TBST and wash the membrane on an incubation shaker for 5 minutes each time, for 4 times.

[0102] ⑦ Color development: The ultrasensitive ECL chemiluminescence detection kit (INNIBIO Yingli Biotechnology Co., Ltd., MF0803-100) was used for color development. The color development result image was obtained using the eBlot electronic tablet imager (Ebit Life Sciences (Shanghai) Co., Ltd.).

[0103] 4) Result determination

[0104] Judgment criteria: If the negative control has no band signal near 15KDa and the positive control has a band signal near 15KDa, and the serum to be tested has a band signal near 15KDa, it is judged as PCMV antibody positive, otherwise it is judged as negative.

[0105] 3. Comprehensive result judgment

[0106] One month after the donor pig is born, four PCMV nucleic acid tests are conducted, and one month before the donor pig is used, two PCMV antibody tests are conducted. If the results are all negative, the pig is considered to be PCMV-negative.

[0107] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0108] Experimental Example 1: Parameter screening for quantitative fluorescence PCR detection of PCMV

[0109] 1. Experimental methods

[0110] (1) Screening of annealing temperature for fluorescence quantitative PCR reaction

[0111] Set different fluorescence quantitative PCR reaction annealing temperatures, test samples of known positive and negative properties, and select the most appropriate annealing temperature based on the results.

[0112] (2) Screening of primer concentration for fluorescence quantitative PCR reaction

[0113] When configuring the PCR reaction system, use different primer concentrations, test samples of known positive and negative properties, and select the most appropriate primer concentration based on the results.

[0114] (3) Screening of detection limits of PCMV antibody Western Blot detection method

[0115] Western Blot was performed to detect porcine cytomegalovirus antibodies by synthesizing viral protein antigens, using the pig serum to be tested as the primary antibody and HRP-labeled rabbit anti-pig IgG antibody as the secondary antibody. In order to understand the detection capability of this method, we serially diluted the samples known to be positive to explore the detection limit of this method.

[0116] 2. Experimental results

[0117] (1) Results of screening for annealing temperature of fluorescence quantitative PCR reaction

[0118] Table 3 Results of screening for annealing temperature of fluorescence quantitative PCR reaction

[0119]

[0120]

[0121] The results (Table 3) showed that false positive results were produced at annealing temperatures of 56°C, 58°C, and 60°C. At annealing temperature of 64°C, samples "positive 5" and "positive 6" with low pathogen copy numbers could not detect the virus. When the annealing temperature was too high, false negative results were produced. Therefore, we believe that 62°C is a relatively ideal annealing temperature.

[0122] (2) Screening of primer concentration for fluorescence quantitative PCR reaction

[0123] Table 4 Results of primer concentration screening for fluorescence quantitative PCR reaction

[0124]

[0125]

[0126] The results (Table 4) show that too high a primer concentration will produce false positive results, and the higher the primer concentration, the higher the false positive rate; while too low a primer concentration will lead to false negative results. According to the analysis of the results, 0.2 μM is a relatively ideal primer concentration.

[0127] (3) Detection limit of PCMV antibody Western Blot detection method

[0128] result( Figure 2 ) showed that using this method, the presence of porcine cytomegalovirus antibodies could still be detected even if the serum was diluted 9600 times, indicating that this method has a high sensitivity.

[0129] The above results show that the optimal annealing temperature of the PCR reaction of the present invention is 62°C, the optimal primer concentration is 0.2 μM, and this method has a high sensitivity and can detect the presence of porcine cytomegalovirus antibodies after the serum is diluted 9600 times.

[0130] In summary, the present invention provides a method for detecting porcine cytomegalovirus. The present invention establishes a porcine cytomegalovirus screening system, and improves the detection limit of porcine cytomegalovirus by optimizing the parameters of quantitative PCR detection. The quantitative PCR in the present invention shortens the detection technology time and has a lower false positive rate; at the same time, the quantitative PCR detection method of the present invention is matched with a PCMV antibody protein immunoblotting detection method, which has a good detection effect for individuals with latent infection and no toxin excretion. The quantitative PCR and protein immunoblotting methods used in the present invention detect porcine cytomegalovirus in medical donor pigs through non-invasive samples, and the result judgment is more intuitive, the false positive rate is lower, the accuracy and sensitivity are high, and it has good application prospects.

Claims

1. A fluorescent quantitative PCR detection method for porcine cytomegalovirus, characterized in that: The method comprises the following steps: (1) extracting DNA from the sample to be tested as a DNA template; (2) performing a PCR reaction: the PCR reaction system includes upstream and downstream primers of porcine cytomegalovirus, a probe and a DNA template; (3) performing fluorescence detection; When the Ct value of the sample to be tested is ≤35.0, the sample to be tested is judged to be positive; when the Ct value of the sample to be tested is ≥40.0, the sample to be tested is judged to be negative; when the Ct value of the sample to be tested is between 35 and 40, adjust the DNA template concentration and repeat the PCR reaction. When the Ct value of the sample to be tested is <40 after the test, the sample to be tested is judged to be positive, and when the Ct value of the sample to be tested is >40 after the test, the sample to be tested is judged to be negative.

2. The fluorescent quantitative PCR detection method according to claim 1, characterized in that: In step (2), the sequences of the upstream and downstream primers of porcine cytomegalovirus are shown as SEQ ID No.1 and SEQ ID No.2 respectively; the sequence of the probe of porcine cytomegalovirus is shown as SEQ ID No.

3.

3. The fluorescent quantitative PCR detection method according to claim 1, characterized in that: In step (2), the PCR reaction system also includes 1× fluorescent quantitative PCR premix and water.

4. The fluorescent quantitative PCR detection method according to claim 1, characterized in that: The final concentrations of the upstream and downstream primers of porcine cytomegalovirus are 0.1-0.8 μmol / L respectively; The final concentration of the porcine cytomegalovirus probe is 0.1-0.8 μmol / L.

5. The fluorescent quantitative PCR detection method according to claim 4, characterized in that: The final concentrations of the upstream and downstream primers of porcine cytomegalovirus are 0.2 μmol / L respectively; the final concentration of the probe of porcine cytomegalovirus is 0.2 μmol / L.

6. The fluorescent quantitative PCR detection method according to claim 1, characterized in that: In step (2), the PCR reaction procedure is: pre-denaturation at 90-100°C for 20-40s; denaturation at 90-100°C for 2-7s, annealing at 56-64°C for 20-40s; 30-45 cycles.

7. The fluorescent quantitative PCR detection method according to claim 6, characterized in that: In step (2), the PCR reaction procedure is: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 62°C for 30 s; 40 cycles.

8. The fluorescent quantitative PCR detection method according to any one of claims 1 to 7, characterized in that: In step (1), the sample to be tested is pig serum or oral and nasal swab sample.

9. A method for detecting porcine cytomegalovirus, characterized in that: It includes the following steps: (i) detecting the porcine cytomegalovirus by the fluorescent quantitative PCR detection method according to any one of claims 1 to 8; (ii) detection using Western blot detection of porcine cytomegalovirus antibodies; If the test results of step (i) and step (ii) are both negative, the sample to be tested is judged to be negative.

10. The method according to claim 9, characterized in that The step (i) is performed in the first month after the donor pig is born, once a week, for a total of 4 times; the step (ii) is performed in the month before the donor pig is used, once every two weeks, for a total of 2 times.

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