Primer and probe for rapidly detecting feline panleucopenia virus through RAA-LFD and application

Abstract

The invention discloses a primer and probe combination for RAA-LFD detection of feline panleucopenia virus and application, and relates to the technical field of molecular biology. According to the primer and probe combination for detecting feline panleucopenia through RAA-LFD, the nucleotide sequence of an upstream primer is shown as SEQ ID NO 3, the nucleotide sequence of a downstream primer is shown as SEQ ID NO 6, and the nucleotide sequence of a probe is shown as SEQ ID NO 9. The whole reaction process is simple to operate and rapid in reaction. The method gets rid of equipment dependence, the requirement for experimental skills of operators in the operation process is simple, the detection time is shortened, the result is convenient to observe, the method is convenient to popularize and use in the grassroots, and the method has huge potential and prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a primer and probe combination and application of RAA-LFD for detecting feline panleukopenia virus. Technical Background

[0002] Feline panleukopenia, also known as feline distemper and feline infectious enteritis, is an acute, highly contagious disease in cats. Feline panleukopenia is a highly contagious disease caused by the feline panleukopenia virus (FPV), which mainly affects cats. The virus belongs to the Parvoviridae family. FPV is widely prevalent and was first discovered in many countries. It is now prevalent worldwide. The virus mainly attacks kittens and adult cats with weakened immunity. Clinical symptoms include high fever, vomiting, diarrhea, loss of appetite, severe dehydration and leukopenia. After infection, cats may have decreased immunity, making them susceptible to other diseases.

[0003] At present, the commonly used laboratory diagnostic methods for feline panleukopenia include electron microscopy, virus isolation, micro HA-HI test, agar diffusion test, micro SN test, PCR test, enzyme-linked immunosorbent assay (ELISA), etc. The traditional isolation and culture method has a long detection cycle, a large workload, and requires high experimental skills of the staff. This experimental operation can only be performed in the laboratory. Immunological diagnostic techniques such as ELISA and immunofluorescence tests have the advantages of low cost and easy operation, but the accuracy needs to be improved. The traditional nucleic acid detection method is PCR technology, but PCR requires a temperature change process, and the results need to be observed by nucleic acid gel electrophoresis and ultraviolet gel imager, so this technology is also limited to the laboratory. In short, these methods are cumbersome to operate and have high requirements for instruments and operators, so they cannot be widely used.

[0004] Recombinase Aided Amplification (RAA) is a new type of nucleic acid isothermal amplification technology that can react under constant temperature conditions. First, the recombinase binds to the primer to form a complex, and then searches for a suitable target site in the double-stranded DNA. After the complex is positioned on the template, the single-stranded binding protein immediately binds to the displaced DNA chain. After the recombinase dissociates, the 3' end of the primer is exposed and recognized by the DNA polymerase. The DNA polymerase begins to amplify the DNA chain at the 3' end of the primer according to the template sequence. At the same time, the nfo enzyme (exonuclease IV) specifically recognizes and cuts THF (tetrahydrofuran), that is, the apurine-free pyrimidine site (AP site), making the blocking group at the 3' end of the probe ineffective, so that the polymerase can continue to extend, and the final amplification product contains both the probe marker and the primer marker. The primer marker specifically binds to the antibody on the test strip to produce a red detection line, and a negative reaction does not produce a red detection line. The application of RAA technology can simplify the experimental process, speed up the detection speed, and improve the convenience and sensitivity of the detection. It is particularly suitable for rapid on-site detection. The establishment and application of rapid detection methods are of great significance for the early diagnosis and timely treatment of feline panleukopenia, which helps to improve the cure rate and reduce the mortality rate, and can more effectively control the spread of the disease and protect the health of the cat population. At present, RAA technology has been applied in many fields, including the rapid diagnosis of animal diseases. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a primer and probe combination for RT-RAA-LFD feline panleukopenia virus, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, and the nucleotide sequence of the probe is shown in SEQ ID NO.9. Further, the probe and primer sequences are modified as follows: the 5' end of the probe is labeled with FA M, a 3C-spacer is introduced at the 3' end for terminal blocking, and the 32nd position of the probe is an abasic site.

[0006] The present invention also provides a kit for detecting feline panleukopenia virus by using RAA-LFD, and the kit comprises the primer and probe combination.

[0007] The present invention also provides a method for detecting feline panleukopenia virus by RAA-LFD, comprising the following steps:

[0008] The genomic DNA of the sample to be tested is used as a template, the primer and probe combination is used to amplify RAA, and the RAA amplification product is detected using a lateral flow chromatography test paper.

[0009] Furthermore, the RAA amplification system is 50 μL, including 29.4 μL of buffer, 2 μL of upstream primer (concentration of 10 μM), 2 μL of downstream primer (concentration of 10 μM), 0.6 μL of probe (concentration of 10 μM), 2.5 μL of magnesium acetate solution, 2 μL of nucleic acid, and the balance is ddH2O.

[0010] Furthermore, the RT-RAA-LFD amplification conditions are a water bath at 32-47° C. for 4-20 min.

[0011] Preferably, the RT-RAA-LFD amplification condition is a reaction at 42° C. in a water bath for 12 min.

[0012] The present invention also provides application of the primer and probe combination in preparing a reagent or a kit for detecting feline panleukopenia virus.

[0013] The beneficial effects of the present invention compared to the prior art are:

[0014] This protocol adopts a simple and fast operation process, with extremely low technical requirements for the experimenter, which significantly simplifies the operation steps. After the RAA reaction is completed, the product only needs to be diluted to quickly obtain the result on the test strip, ensuring the rapidity and accuracy of the test. The entire detection process takes no more than 30 minutes, which greatly shortens the operation time compared with traditional detection technology. The detection method developed in this protocol has extremely high specificity, only produces a positive reaction to feline panleukopenia virus, and shows negative results for other common cat pathogens or pathogens of similar genotypes, ensuring high accuracy of the test. It also has extremely high sensitivity and can detect as low as 10 copies of nucleic acid, which is 10 times the sensitivity of real-time fluorescence PCR technology. At the same time, the detection method of this protocol is free from dependence on the laboratory environment. RAA technology can react under constant temperature conditions, and only a water bath is required to complete the entire process. In addition, the reaction product is directly observed through the test strip, without the need for nucleic acid gel electrophoresis and UV gel imager analysis. These measures significantly reduce the cost of detection and are more suitable for large-scale detection at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is a schematic diagram of the screening of primers and probes for FCV detection, wherein A is a schematic diagram of the screening of test strips, and B is a schematic diagram of the screening of gel electrophoresis;

[0016] Figure 2 Schematic diagram of RAA-LFD temperature optimization;

[0017] Figure 3 Schematic diagram of RAA-LFD time optimization;

[0018] Figure 4Schematic diagram of RAA-LFD specific detection;

[0019] Figure 5 Schematic diagram of RAA-LFD sensitivity detection and its gel electrophoresis diagram, wherein A is the sensitivity detection diagram and B is the gel electrophoresis screening diagram;

[0020] Figure 6 is the sensitivity of real-time fluorescence PCR, where 1 to 7 are the plasmid concentrations of 1×10 7 ~1×10 copies / uL, 8 as negative control;

[0021] Figure 7 Schematic diagram of RAA-LFD repeatability detection. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0023] In the following examples, if the experimental methods are not specifically specified, they are generally carried out according to conventional experimental conditions or the conditions recommended by the reagent manufacturers. Unless otherwise specified, all reagents and materials are obtained from commercial sources.

[0024] Example 1: Amplification of viral target sequences and design and screening of RAA primer and probe combinations

[0025] 1. Amplification of viral target sequence

[0026] Extract feline panleukopenia virus nucleic acid, and design PCR primers CPV-F (SEQ ID NO.1: CTACTCAGCCACCAACTA) and CPV-R (SEQ ID NO.2: TTCCCATTTGAGTTACACC) according to the VP2 gene sequence to amplify the VP2 gene. Reaction system: VP2-F (10 μM) 1 μL, VP2-R (10 μM) 1 μL, nucleic acid 4 μL, add dd H2O to 50 μL. Reaction conditions are as follows: 94℃1min; 94℃10s, 55℃10s, 72℃30s, 35 cycles; 72℃7min. Detect PCR products by gel electrophoresis, recover the target fragments by gel and purify.

[0027] The target fragment and vector pET-30a(+) were digested with restriction endonucleases, and the target fragment after digestion was connected to the vector at 16°C for 12 hours. Take 5 μL of the ligation product and transform it into DH5α competent cells, and screen the clones that successfully inserted the target fragment for expansion culture. The plasmid was extracted with a plasmid mini-extraction kit and digested for identification. The correctly identified recombinant plasmid was sent to Shanghai Biotech for sequencing. The sequencing results have high homology with the VP2 gene sequence fragment, so the plasmid of the target fragment of the VP2 gene sequence was obtained. According to Moore's law, the DNA copy number = 6.02×10 23 ×Plasmid concentration (ng / μL)×10 -9 / (660×standard plasmid base number) to calculate the number of DNA copies contained in unit volume of plasmid.

[0028] 2. Design and screening of RAA primers and probes:

[0029] As of March 2024, the FPV full genome sequence was downloaded from GenBank in my country, and the genome sequence was aligned using DNASTAR (DNASTAR Inc., Madison, WI, USA) software to determine the conserved region of the FPV genome. A probe was designed using Oligo7 software, and three sets of corresponding primers (FPV-F1 / R1, FPV-F2 / R2, and FPV-F3 / R3) were designed upstream and downstream of the probe. In order to improve the binding efficiency of the probe, the Tm value of the probe was designed to be 10°C higher than that of the primer. It was synthesized by Shanghai Bioengineering Co., Ltd. The sequence is shown in Table 1:

[0030] Table 1

[0031]

[0032]

[0033] Note: Biotin: biotin; FAM: 6-carboxyfluorescein; THF: tetrahydrofuran residue; C3 Spacer: blocking substance.

[0034] Primer design principles: 1. Primer length is 30-35bp; 2. Primer GC content is 20%-70%; 3. Primer Tm value is 50-100; 4. The 5' end of the downstream primer is labeled with biotin; 5. The amplification product is 100-200bp.

[0035] Probe design principles: 1. The probe length is at least 46bp; 2. The 5' end of the probe is labeled with FAM, and a 3C-spacer is introduced at the 3' end for end blocking; 3. Tetrahydrofuran THF is introduced in the middle of the probe, that is, a purine-free and pyrimidine-free site (AP site) as the recognition site of the nfo enzyme. At least 30bp of bases must be retained before the AP site, and at least 15bp of bases must be retained after the AP site. The amplified product has FAM at one end and biotin at the other end. Biotin specifically binds to the antibody on the test strip, causing a red line to appear on the test line, which is different from a negative reaction, so it can be detected by LFD. The above-designed and synthesized primer probe sets were used for RAA amplification, reacted at 40°C for 12 minutes, and FCV nucleic acid was extracted as a template as a positive control (PC).

[0036] Take part of the reaction product, dilute it and use the test strip for detection. The positive amplification product has FAM on one end and biotin on the other end. Biotin specifically binds to the antibody on the test strip, making a red line appear on the test line. Conversely, if there is no amplification product in the negative reaction, no test line will appear. Figure 1 As shown in (A), the detection line of group F1 / R1 / P1 is the darkest, indicating the most amplified products and the best amplification effect.

[0037] At the same time, part of the product was purified and subjected to agarose gel electrophoresis, and the best primer probe set was screened according to the brightness of the target band. Figure 1 As shown in (B), the bands of the F1 / R1 / P1 group were the brightest, indicating that its amplification effect was the best.

[0038] In summary, FCV-F1 / R1 / P1 is the best primer-probe set.

[0039] Example 2: RAA-LFD temperature and time optimization:

[0040] 1. RAA-LFD temperature optimization

[0041] Use pET-30a-VP2 plasmid as template and designed primer probe to configure reaction solution for amplification. Amplification conditions are set to gradient temperature: 32°C, 37°C, 42°C, 47°C, 52°C. Put the configured reaction solution into the corresponding temperature for reaction. Set blank control NTC and reaction time to 15 minutes. After the reaction is completed, take 5μL of reaction product and dilute it 20 times with dd H2O. Take 50μL of dilution and add it to the sample area of ​​the test strip. Filter out the best reaction temperature according to the detection line. Figure 2 As shown, the best detection result is when the reaction temperature is between 32℃ and 47℃; the detection line color is darkest when the reaction temperature is 42℃; when the reaction temperature is 38℃, the temperature is low and the reaction is not sufficient, and the product is less; when the reaction temperature is 52℃, the temperature is high and the enzyme loses its activity.

[0042] 2. RAA-LFD time optimization

[0043] Use pET-30a-VP2 plasmid as template and designed primer probe to configure reaction solution for amplification. Set the amplification temperature to 42°C, and set the gradient reaction time to 4min, 8min, 12min, 16min, and 20min. Place the configured reaction solution for the corresponding reaction time, and set a blank control NTC. After the reaction is completed, draw 5μL of the reaction product and dilute it 20 times with dd H2O, draw 50μL of the dilution solution and add it to the sample area of ​​the test strip, and screen out the best reaction time according to the detection line. Figure 3 As shown, the detection system can detect the result within 8 minutes of reaction, but the color of the detection line is weak; the color of the detection line is darkest after 12 minutes of reaction, and 12 minutes is selected as the best reaction time.

[0044] Example 3: RAA-LFD specific detection

[0045] The RAA-LFD detection method is used to detect nucleic acids of common cat pathogens, namely feline herpesvirus (FHV), feline parvovirus (FPV), feline calicivirus (FCV), feline coronavirus (FCoV), and feline Salmonella. Specific steps:

[0046] Extract nucleic acid of FHV, FPV, FCV, FCoV, and Salmonella;

[0047] RAA-LFD primer probe was used to detect these five nucleic acids. The reaction system was 29.4μL Buffer A, 2μL upstream primer, 2μL downstream primer, 0.6μL probe, 2.5μL Buffer B, 2μL nucleic acid, and ddH2O was added to make up to 50μL. The reaction was carried out at 42℃ for 12 minutes. After the reaction was completed, 5μL of the reaction product was aspirated and diluted 20 times with dd H2O. 50μL of the dilution was aspirated and added to the sample area of ​​the test strip. If a red detection line appeared, it was positive. If not, it was negative. Figure 4 As shown in the figure, only feline parvovirus nucleic acid was positive. The results showed that the RAA-LFD detection method had good specificity.

[0048] Example 4: RAA-LFD sensitivity detection

[0049] 1. Template preparation

[0050] The extracted plasmid containing the target fragment of VP2 gene sequence was diluted to a gradient concentration of 1.0×10 8 copies / μL~1.0×10 0 copies / μL.

[0051] 2. RAA-LFD detection

[0052] RAA-LFD reaction was performed using gradient concentration plasmid as template. The reaction system included 29.4 μL buffer, 2 μL upstream primer, 2 μL downstream primer, 0.6 μL probe, 2.5 μL magnesium acetate solution, 2 μL nucleic acid, and ddH2O to make up to 50 μL. The reaction was carried out at 42°C for 8 minutes. After the reaction was completed, 5 μL of the reaction product was aspirated and diluted 20 times with sterile water. 50 μL of the dilution was aspirated and added to the sample area of ​​the test strip. If a red test line appeared, it was positive. If not, it was negative. Figure 5 As shown in (A), RAA-LFD can detect concentrations as low as 1.0×10 1 copies / μL of plasmid.

[0053] 3. PCR-electrophoresis detection

[0054] The PCR reaction was carried out using the plasmid with gradient concentration as template, the primers were the upstream and downstream primers of RAA, and the product length was 158bp. Reaction system: PCR SuperMix for PAGE (+dye) (2×) 25μL, upstream primer 1μL, upstream primer 1μL, nucleic acid 2μL, add sterile water to 50μL. The reaction conditions are as follows: 94℃1min; 94℃10s, 55℃10s, 72℃10s, 35 cycles; 72℃7min. The reaction product was subjected to nucleic acid gel electrophoresis at 120V for 30 minutes, and the detection results were observed with a UV gel imager. Figure 5 As shown in (B), the minimum detection limit of PCR-electrophoresis detection is 1.0×10 3 copies / μL of plasmid.

[0055] 4. Real-time fluorescence PCR detection

[0056] The RAA test was paralleled using the real-time fluorescence PCR test method published in Jilin Provincial Standard DB22 / T 2788-2017. The primer and probe sequences (5'→3') of the method in this standard are as follows:

[0057] Upstream primer F (SEQ ID NO. 9): 5'-AATGCTTGGGGAGTTTGGTTT-3'.

[0058] Downstream primer R (SEQ ID NO. 10): 5'-TTTAGTTGGTGGCTGAGTAGCAGA-3'.

[0059] The PCR reaction was performed using the plasmid with gradient concentration as the template. The reaction system: SYBR Premix Ex Tag II (2×) 25 μL, upstream primer 1 μL, upstream primer 1 μL, nucleic acid 2 μL, add dd H2O to make up to 50 μL. The reaction conditions were as follows: 95℃30s; 95℃5s, 60℃30s, 40 cycles. Figure 6 The lowest detection limit of the fluorescent PCR was 1.0 × 10 2 of the plasmid.

[0060] In summary, the minimum detection limits of RAA-LFD detection, PCR-electrophoresis detection, and fluorescence PCR detection were 1.0×10 1 copies / μL, 1.0×10 3 copies / μL, 1.0×10 2 copies / μL, the RAA-LFD detection method has higher sensitivity.

[0061] 5. Repeatability test

[0062] To test the repeatability of the FPV RAA-LFD method, the constructed plasmid standard was used as a template for the reaction. Figure 7 The results showed that the method had good repeatability, and the minimum detection rate after three repeated tests was 1.0×10 1 copies / μL.

[0063] Example 5: Detection of clinical samples

[0064] 72 tissue samples and nasal swabs of the diseased cats were taken, and their nucleic acids were extracted and tested using the RAA-LFD detection method. The test results were also compared with the fluorescent PCR method. Among the 72 cat tissue samples, RAA-LFD detected 19 positive results, with a consistency of 98.5% with traditional PCR, and the sensitivity was higher than conventional PCR. The comparison of the clinical test results of RAA-LFD and the PCR results is shown in Table 2.

[0065] Table 2

[0066]

[0067] In summary, the consistency between the RAA-LFD detection method and the PCR detection results is 98.5%. The RAA-LFD method created by the present invention is an efficient, rapid and portable method for detecting feline panleukopenia virus, which provides important technical support for on-site detection of feline pathogens.

[0068] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A primer and probe combination for detecting feline panleukopenia virus by RAA-LFD, characterized in that: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, and the nucleotide sequence of the probe is shown in SEQ ID NO.

9.

2. The primer and probe combination according to claim 1, characterized in that: The probe and primer sequences were modified as follows: the 5' end of the probe was labeled with FAM, a 3C-spacer was introduced into the 3' end for terminal blocking, and the 32nd position of the probe was an abasic site.

3. A kit for detecting feline panleukopenia virus by RAA-LFD, characterized in that: The kit comprises the primer and probe combination of claim 1.

4. A method for detecting feline panleukopenia virus using RAA-LFD, characterized in that: The steps include: The genomic DNA of the sample to be tested is used as a template, the primer and probe combination of claim 1 is used to amplify RAA, and the RAA amplification product is detected using a lateral flow chromatography test paper.

5. The method according to claim 4, characterized in that The RAA amplification system is 50 μL, including 29.4 μL of buffer, 2 μL of upstream primer (concentration of 10 μM), 2 μL of downstream primer (concentration of 10 μM), 0.6 μL of probe (concentration of 10 μM), 2.5 μL of magnesium acetate solution, 2 μL of nucleic acid, and the balance is ddH2O.

6. The method according to any one of claims 4 or 5, characterized in that: The RT-RAA-LFD amplification conditions are as follows: reacting in a water bath at 32-47° C. for 4-20 min.

7. The method according to claim 6, characterized in that The RT-RAA-LFD amplification condition is to react in a water bath at 42° C. for 12 min.

8. Use of the primer and probe combination according to claim 1 or 2 in preparing a reagent or a kit for detecting feline panleukopenia virus.

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