Primer probe combinations, kits and methods for detecting crocodilepox virus

Abstract

The application belongs to the field of biological detection, and particularly relates to a primer probe combination for detecting crocodile pox virus, which comprises a forward primer, a reverse primer and a probe; the sequence of the forward primer is shown as SEQ ID NO:1, the sequence of the reverse primer is shown as SEQ ID NO:2, and the sequence of the probe is shown as SEQ ID NO:3. The primer probe combination can quickly and accurately detect the crocodile pox virus in a sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and particularly relates to a primer probe combination, a kit and a method for detecting crocodile poxvirus. BACKGROUND

[0002] Poxviridae is a double-stranded DNA virus, which is the most complex and largest virus group known at present, and can infect a variety of hosts including vertebrates and invertebrates. Crocodile poxvirus belongs to the genus Crocodylidpoxvirus under the subfamily Chordopoxvirinae, and mainly infects reptiles such as crocodiles. The virus can be transmitted between different reptiles, causing severe skin lesions, and the clinical manifestations include skin rash, hyperkeratosis, and various skin damage from wart-like lesions to deep penetrating epidermal cysts. Ulcers are also commonly reported around the mouth, eyes and nostrils. In the breeding environment, factors such as high-density breeding and insufficient nutrition can significantly increase the risk of virus transmission. In addition, some infected individuals show asymptomatic carriage, which becomes a potential source of hidden infection, leading to the persistence and spread of the virus in the breeding farm.

[0003] China is the largest alligator farming country in the world, and the alligator farming industry has developed rapidly in recent years. According to the data of the “2024-2030 China Alligator Farming Industry Market Status Survey and Development Trend Judgment Report”, as of 2018, the number of alligator farmers in China reached 36,000, with a breeding area of about 145 million mu and a total breeding amount of 390 million, accounting for more than 70% of the global total. Although the mortality rate of alligators infected with poxvirus is not high, the incidence rate is high, and the skin damage caused by the virus seriously affects the value of alligator skin, causing significant economic losses to the breeding industry. Therefore, it is of great significance to establish effective prevention and control measures in the breeding process.

[0004] With the progress of molecular biology technology, PCR technology has become an important tool for pathogen detection due to its rapidness, efficiency and other characteristics. Among them, real-time fluorescent quantitative PCR can realize the accurate quantification of target nucleic acid by monitoring the PCR amplification process in real time through fluorescence signal. Compared with traditional methods, this technology greatly shortens the detection time, reduces the cost of manpower and material resources, and provides reliable support for early diagnosis of pathogens. However, there is still a lack of real-time fluorescent PCR detection products specifically for crocodile poxvirus in the current public technology.

[0005] Under this background, there is an urgent need in the field to develop a reagent and method for rapidly and accurately detecting crocodile poxvirus to meet the detection needs of the breeding industry for high specificity, high sensitivity, good reproducibility and short detection period. SUMMARY

[0006] In order to solve the problems in the background art, the purpose of the present application is to provide a primer probe combination for detecting crocodile poxvirus, which can quickly and accurately detect crocodile poxvirus in a sample to be tested.

[0007] The solution adopted by the present application to solve the technical problems is a primer probe combination for detecting crocodile poxvirus, which comprises a forward primer, a reverse primer and a probe; the sequence of the forward primer is shown in SEQ ID NO: 1, the sequence of the reverse primer is shown in SEQ ID NO: 2, and the sequence of the probe is shown in SEQ ID NO: 3.

[0008] Further, the probe is modified at both ends with a fluorescent reporter group and a fluorescent quencher group, respectively.

[0009] The fluorescent reporter group is selected from any one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED460, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

[0010] The second purpose of the present application is to provide the use of the primer probe combination in the preparation of a kit for detecting crocodile poxvirus.

[0011] The third purpose of the present application is to provide a kit for detecting crocodile poxvirus, which comprises PCR amplification reagents, a negative control and a positive control, wherein the PCR amplification reagents comprise a primer probe combination; the primer probe combination comprises a forward primer with a sequence shown in SEQ ID NO: 1, a reverse primer with a sequence shown in SEQ ID NO: 2, and a probe with a sequence shown in SEQ ID NO: 3.

[0012] Preferably, the PCR amplification reagents further comprise a DNA polymerase, Mg 2+ , dNTP.

[0013] Preferably, in the primer probe combination, the concentrations of the forward primer, the reverse primer and the probe are all 10 μM.

[0014] Preferably, the positive control is a plasmid containing a fragment of crocodile poxvirus, and the negative control is ultrapure water.

[0015] The fourth purpose of the present application is to provide a method for detecting crocodile poxvirus for non-diagnostic treatment purposes, comprising the following steps:

[0016] 1) extracting total DNA from a sample to be tested;

[0017] 2) preparing a reaction system, the reaction system comprising PCR amplification reagents, the PCR amplification reagents comprising a primer probe combination; the primer probe combination comprising a forward primer with the sequence shown in SEQ ID NO: 1, a reverse primer with the sequence shown in SEQ ID NO: 2, and a probe with the sequence shown in SEQ ID NO: 3;

[0018] 3) performing real-time fluorescent quantitative PCR reaction by taking the extracted total DNA of the sample to be tested as a template, and adding negative control and positive control as quality control templates;

[0019] 4) determining the detection result according to the Ct value after the reaction is completed.

[0020] Preferably, the program of the real-time fluorescent quantitative PCR reaction is: 94℃, 5min; 94℃, 10s, 60℃, 30s, performing 40 cycles.

[0021] In summary, the application provides a crocodile poxvirus primer probe combination, kit and detection method for real-time fluorescent quantitative PCR detection. The scheme can quickly and accurately detect crocodile poxvirus in the sample to be tested, and has no cross reaction with other pathogens, and can effectively distinguish other poxviruses. The method has high sensitivity, strong specificity, good practicability, and the detection lower limit can reach 1000 copies / mL, and can realize specific detection of crocodile poxvirus in the early stage of infection, and is suitable for timely screening and prevention and control of the pathogen.

[0022] The above description is only a summary of the technical solutions of the application, in order to more clearly understand the technical means of the application, the content of the specification can be implemented, and in order to make the above and other purposes, characteristics and advantages of the application more obvious and easy to understand, the following preferred embodiments are described in detail below, and the accompanying drawings are described as follows. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For experiment one, the detection of plasmid linearity is shown in the machine result graph.

[0024] Figure 2 For experiment one, the standard curve graph of detecting plasmid linearity is shown.

[0025] Figure 3 For experiment three, the machine result graph of detecting positive samples is shown. DETAILED DESCRIPTION

[0026] In order to make the content of the application more easily and clearly understood, the following describes the application according to specific embodiments and in conjunction with the drawings.

[0027] Experiment one

[0028] According to the nucleotide sequence registered in GenBank, Saltwater crocodilepox virus (accession number MK903855.1) and Nile crocodilepox virus (accession number NC 008030.1) were selected for bioinformatics software alignment analysis, and the conservative region was selected as the amplification target sequence region, and the alligatorpox virus plasmid was synthesized. Three pairs of primer probe combinations (labeled as T1, T2 and T3, respectively) were designed by using primer probe design software Primer Premier 5.0 and oligo 7, and the plasmid was amplified, and a pair of certain primer probe combination was selected through experiment.

[0029] The specific screening process is as follows: the sequences of the three pairs of primer probe combinations are shown in Table 1, and each primer probe is diluted to 10 M concentration, and the PCR amplification reagent is prepared according to Table 2; the synthesized alligatorpox virus plasmid is diluted after setting value, and diluted to 1×10 4 Copies / mL and 1×10 8 Copies / mL. T1, T2 and T3 are added with 5 L concentration plasmid for amplification.

[0030] Table 1 Primer probe sequence information

[0031]

[0032] Table 2 PCR amplification system

[0033]

[0034] Table 3 Linear test results

[0035]

[0036] Experimental results

[0037] The linear test results of the plasmids of T1, T2 and T3 are shown in Table 3, and the on-machine results of the plasmid linearity are shown in Figure 1 The comparative analysis shows that the performance of T1 primer probe combination is better than that of T2 and T3 in terms of fluorescence intensity and sensitivity index. Further, the experimental data of T1 combination are used to draw a plasmid linear detection standard curve, and the results are shown in Figure 2 The combination has an amplification efficiency of 98.397% in real-time fluorescent PCR, and the determination coefficient (R²) is 1.0000, and the amplification effect is good, which is suitable for subsequent quantitative detection.

[0038] Therefore, F1 / R1 / PF1 is selected as the final primer probe:

[0039] Forward primer (CRV F1): 5'-ACGTCATCTCGTCGTCGATC-3', as shown in SEQ ID NO: 1;

[0040] Reverse primer (CRV R1): 5'-CGTCGAAGTCCGCGTTCTG-3', as shown in SEQ ID NO: 2;

[0041] Probe (CRV PF1): 5'-CGCGACGGCCGAGAACACGA-3', as shown in SEQ ID NO: 3.

[0042] The 5' end of the probe of the present embodiment is labeled with a fluorescent reporter group FAM, and the 3' end of the probe is labeled with a fluorescent quencher group BHQ1. The amplification length of the forward primer, the reverse primer and the probe is 67 bp.

[0043] Experiment two

[0044] Based on the results of experiment one, the T1 primer probe combination screened was used to analyze the performance of the plasmid standard and the clinical sample, and the specific content is as follows:

[0045] 1) Reproducibility test

[0046] The synthesized alligatorpox virus plasmid was diluted to 1x10 5 copies / mL and 1x10 7 copies / mL. The PCR amplification reagent was prepared according to Table 2, 5 L of each alligatorpox virus plasmid concentration group was taken into the PCR amplification reagent for amplification, and each was repeated 20 times. The reproducibility test results are shown in Table 4.

[0047] Table 4 Reproducibility test results

[0048]

[0049] As shown in Table 4, the coefficient of variation of the primer probe combination and the detection method provided by the present application in the 1x10 5 copies / mL and 1x10 7 copies / mL plasmid groups were 1.14% and 0.83% respectively, CV<2%, and the reproducibility was good.

[0050] 2) Sensitivity test

[0051] The synthesized alligatorpox virus plasmid was diluted to 1x10 3 copies / mL and 5x10 2Copies / mL. Prepare PCR amplification reagents according to Table 2, and take 5 copies / mL of each crocodile poxvirus plasmid concentration group for amplification into the PCR amplification reagent, repeat 20 times, and the repeatability test results are shown in Table 5.

[0052] Table 5 Sensitivity test results

[0053]

[0054] As shown in Table 5, the primer probe combination and the detection method provided by the application can detect 1x10 3 copies / mL with a detection rate of 100%, and 5x10 2 copies / mL with a detection rate of 75%. Therefore, the sensitivity of the crocodile poxvirus is 1x10 3 copies / mL.

[0055] Summary

[0056] In summary, according to Figures 1-2 the detection results shown in Tables 3-5, it can be seen that the T1 primer probe combination and the detection method provided by the application have a detection limit of 1000 copies / mL, and have the characteristics of short required time, good repeatability, and high sensitivity.

[0057] Experiment three

[0058] This example shows a kit for detecting crocodile poxvirus based on real-time fluorescent quantitative PCR technology, including PCR amplification reagent, negative control and positive control. Among them, the PCR amplification reagent includes the primer probe combination for detecting crocodile poxvirus as shown in SEQ ID NO: 1-3 and 2x Buffer Mix (DNA polymerase, Mg 2+ , dNTP), the positive control is an artificially synthesized plasmid containing the target fragment of crocodile poxvirus, and the negative control is ultrapure water. Among them, the concentrations of the forward primer, the reverse primer and the probe are all 10 M, and the PCR amplification reagent is shown in Table 1. The specific operation steps are as follows:

[0059] 1) Extract the nucleic acid of the sample to be tested; place the collected skin lesion swab in the sample preservation liquid, mix thoroughly, and then take 200 l to the lysis solution of the Juzhong Medical nucleic acid extraction reagent consumables (cassette), and use the Juzhong Medical nucleic acid extractor for nucleic acid extraction.

[0060] 2) Prepare the PCR amplification reagent according to Table 2, and take 5 l of the sample nucleic acid to be tested, the positive control, and the negative control into the PCR amplification reagent. Perform real-time fluorescent quantitative PCR reaction according to the following program;

[0061] Step1: 94℃, 5min;

[0062] Step2: 94℃, 10s, 60℃, 30s;

[0063] Step2 performs 40 cycles.

[0064] 3) After the reaction is completed, the detection result is determined according to the Ct value.

[0065] When determining, the fluorescence curve above the threshold value should be a clear S-shaped curve, otherwise the experiment is invalid and the instrument, reagent, amplification condition and the like should be checked.

[0066] When detecting the crocodile poxvirus by the method provided in the application, the judgment standard is as follows:

[0067] Under the condition that the negative control has no amplification and the Ct value of the positive control is less than 35, the Ct of the sample to be tested is less than or equal to 38, which is determined as positive. If the Ct value of the sample to be tested is 38 < Ct ≤ 40, it is recommended to retest, and the Ct value of the retest result is less than or equal to 40, which is determined as positive, otherwise it is negative.

[0068] The detection result is shown in Table 6. Figure 3 and Table 6, it can be seen that the primer probe combination, detection kit and detection method provided in the application can efficiently and accurately detect the crocodile poxvirus in the sample to be tested.

[0069] Table 6: Sample detection results

[0070]

[0071] In summary, the above embodiments are only preferred embodiments of the application and do not limit the protection scope of the application, and any modification, equivalent replacement, improvement and the like made within the spirit and principle of the application shall be included in the protection scope of the application.

[0072] In addition, it should be noted that the fluorescence reporter group in the system can also be replaced or selected as one of VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED460, and the quenching group can also be replaced or selected as one of BHQ2, BHQ3 or MGB according to the actual application.

[0073] The above-described embodiments are only preferred embodiments of the application and cannot limit the protection scope of the application, and any non-substantial changes and modifications made by those skilled in the art on the basis of the application shall be included in the protection scope of the application.

Claims

1. A primer probe combination for detecting crocodilepoxvirus, characterized by, The primer probe combination comprises a forward primer, a reverse primer and a probe; the sequence of the forward primer is shown as SEQ ID NO: 1, the sequence of the reverse primer is shown as SEQ ID NO: 2, and the sequence of the probe is shown as SEQ ID NO:

3.

2. The primer probe combination for detecting crocodilepoxvirus according to claim 1, wherein, The probe is modified with a fluorescent reporter group and a fluorescent quencher group at both ends thereof; The fluorescent reporter group is selected from any one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED460, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

3. Use of the primer probe combination of any one of claims 1 or 2 in the preparation of a crocodilepox virus detection kit.

4. A kit for detecting crocodilepoxvirus, characterized by, The kit comprises PCR amplification reagents, a negative control and a positive control, wherein the PCR amplification reagents comprise a primer probe combination; the primer probe combination comprises a forward primer with the sequence shown as SEQ ID NO: 1, a reverse primer with the sequence shown as SEQ ID NO: 2, and a probe with the sequence shown as SEQ ID NO:

3.

5. The kit for detecting crocodilepoxvirus according to claim 4, characterized in that, The PCR amplification reagent further includes a DNA polymerase, Mg 2+ , dNTP.

6. The kit for detecting crocodilepox virus according to claim 4, characterized in that, In the primer probe combination, the concentrations of the forward primer, the reverse primer and the probe are all 10 μM.

7. The kit for detecting crocodilepox virus according to claim 4, characterized in that, The positive control is a plasmid comprising a crocodilepox virus target fragment, and the negative control is ultrapure water.

8. A method for detecting crocodilepoxvirus for non-diagnostic therapeutic purposes, characterized in that, The kit comprises the following steps: 1) extracting total DNA of a sample to be tested; 2) preparing a reaction system, wherein the reaction system comprises PCR amplification reagents, and the PCR amplification reagents comprise a primer probe combination; the primer probe combination comprises a forward primer with the sequence shown as SEQ ID NO: 1, a reverse primer with the sequence shown as SEQ ID NO: 2, and a probe with the sequence shown as SEQ ID NO: 3; 3) performing real-time fluorescent quantitative PCR reaction by using the extracted total DNA of the sample to be tested as a template, and adding a negative control and a positive control as quality control templates; 4) determining the detection result according to the Ct value after the reaction is completed.

9. The method of claim 8, wherein the detection of crocodilepoxvirus is for non-diagnostic therapeutic purposes. The program of the real-time fluorescent quantitative PCR reaction is: 94℃, 5min; 94℃, 10s, 60℃, 30s, performing 40 cycles.

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