CN113373266B - Fluorescent RPA primer, kit and detection method for detecting sheep pulmonary adenomatosis virus - Google Patents
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Abstract
The invention discloses a fluorescent RPA primer, a kit and a detection method for detecting sheep pulmonary adenomatosis virus, and relates to the technical field of virus detection. The invention provides a novel fluorescent RPA composition for detecting sheep pulmonary adenomatosis virus, which comprises the following primers and a probe matched with the primers: the forward primer is a nucleotide sequence shown as SEQ ID No. 3; the reverse primer is a nucleotide sequence shown as SEQ ID No. 4; the probe is a nucleotide sequence shown as SEQ ID No. 11. The composition can be used for efficiently and quickly detecting whether a sample contains the sheep pulmonary adenomatosis virus or not based on a recombinase polymerase amplification technology, and has high detection sensitivity and strong specificity. The invention also provides a fluorescent RPA kit for detecting the sheep pulmonary adenomatosis virus and a method for detecting the sheep pulmonary adenomatosis virus by using the fluorescent RPA composition.
Description
Technical Field
The invention relates to the technical field of virus detection. More particularly, relates to a fluorescent RPA primer, a kit and a detection method for detecting sheep pulmonary adenomatosis virus.
Background
Sheep Pulmonary adenomatosis (OPA) is a chronic, contact, infectious, Pulmonary neoplastic disease caused by Exogenous Sheep Pulmonary adenomatosis virus (exJSRV). Is mainly characterized by cough, dyspnea, emaciation, massive serous rhinorrhea, and tumorous hyperplasia of II-type alveolar epithelial cells and bronchiolar epithelial cells of the affected sheep. OPA cases have been identified in different breeds of sheep in several national regions of the world. Because the clinical characteristics of the disease are not obvious, the latent period is long, the infection is not easy to be found, the epidemic disease spreads in a large scale and is vertically spread. The diseased mutton sheep are progressively thinned and finally died in a large scale, and the diseased breeding sheep are low in hybridization rate and finally eliminated. The first cloned sheep in the world also died of Dolly. Thus, OPA is of great interest as an important economic and animal welfare issue. Since sheep pulmonary adenomatosis mainly infects sheep of various varieties and different ages, especially expensive breeding sheep are more susceptible. Once sheep are infected with the virus, the fatality rate is 100%. Therefore, the continuous existence of OPA seriously threatens the production of breeding sheep and the development of sheep raising industry, and simultaneously threatens the food safety of mutton, thereby causing great economic loss.
At present, no in-vitro culture environment suitable for JSRV propagation is established, so that the JSRV can not be diagnosed by adopting the existing virus identification and separation method. Meanwhile, enJSRVEv (endogenous sheep pulmonary adenomatosis virus) is expressed in sheep in large quantity, and sheep cannot form immune tolerance to exJSRV so that specific antibodies cannot be detected in the sheep in vivo, so OPA cannot be diagnosed by serological tests. At present, the diagnosis of exJSRV in a laboratory mainly comprises detection methods such as PCR, dot hybridization technology, ELISA, LAMP and the like. However, the PCR detection technology is complex to operate, needs professional technicians and needs 1-2 hours to see a detection result, cannot be popularized in the basic level, and cannot realize the on-site rapid detection of pathogens; although the dot blot hybridization technology can be used for clinical diagnosis, the result can be detected only in 2-3 days, the reaction temperature is required to be changed in the detection process, and the operation is relatively complex; ELISA has high cost and complex operation, and is not suitable for large-scale detection of farms; the LAMP detection process needs 6 primers to be completed, so the primer design of the method is quite complex, and false positive results are easy to appear because the test tube needs to be opened when the result is judged.
Therefore, a kit for detecting the sheep pulmonary adenomatosis virus, which is rapid, highly sensitive and highly specific, needs to be developed to realize rapid detection of the sheep pulmonary adenomatosis virus and complete field diagnosis outside a laboratory.
Disclosure of Invention
In view of the above problems, the first objective of the present invention is to provide a set of fluorescent RPA compositions for detecting ovine pulmonary adenomatosis virus, by which whether a sample contains ovine pulmonary adenomatosis virus can be detected efficiently and rapidly based on recombinase polymerase amplification technology, and the detection sensitivity and specificity are high.
The second purpose of the invention is to provide a fluorescent RPA kit for detecting sheep pulmonary adenomatosis virus.
It is a third object of the present invention to provide a method for detecting ovine pulmonary adenomatosis virus using the fluorescent RPA compositions of the present invention.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a fluorescent RPA composition for detecting sheep pulmonary adenomatosis virus, which comprises the following primers and a probe matched with the primers:
the forward primer is a nucleotide sequence shown as SEQ ID No. 3;
the reverse primer is a nucleotide sequence shown as SEQ ID No. 4;
the probe is a nucleotide sequence shown as SEQ ID No. 11.
Preferably, the probe is substituted with THF at the 31 st base "a" from the 5 'end, with the T base upstream of the THF site labeling the FAM fluorophore, the T base downstream of the THF site labeling the BHQ1 fluorophore, and the C3 Spacer at the 3' end.
The invention also provides application of the fluorescent RPA composition in preparing a fluorescent RPA kit for detecting sheep pulmonary adenomatosis virus.
According to a second object of the present invention, the present invention also provides a fluorescent RPA kit for the detection of ovine pulmonary adenomatosis virus, said kit comprising a fluorescent RPA composition as described above.
Preferably, the kit further comprises a positive control.
Preferably, the positive control is in vitro reverse transcribed cDNA of the ovine pulmonary adenovirus env gene.
Preferably, the kit further comprises a recombinase for binding the single-stranded nucleic acid, a single-stranded DNA binding protein, a strand displacement DNA polymerase, a reaction system buffer and magnesium acetate.
According to a third object of the present invention, the present invention further provides a method for detecting ovine pulmonary adenomatosis virus, the method comprising:
extracting total RNA of a sample to be detected, and performing reverse transcription to obtain cDNA;
formulating a fluorescent RPA reaction system comprising the fluorescent RPA composition of claim 1 or 2;
carrying out fluorescent RPA amplification reaction by taking cDNA obtained by reverse transcription as a template;
and (6) judging the result.
Preferably, the fluorescent RPA reaction system comprises 2.1. mu.L of 10. mu.M forward primer, 2.1. mu.L of 10. mu.M reverse primer and 0.6. mu.L of 10. mu.M probe, wherein the total volume is 50. mu.L.
Preferably, the reaction temperature of the fluorescent RPA amplification reaction is 40 ℃ and the reaction time is 20 min.
The invention has the following beneficial effects:
the fluorescent RPA composition and the method for detecting sheep pulmonary adenomatosis virus by using the fluorescent RPA composition are designed according to exJSRV conserved Env gene which can code Env playing a main carcinogenic role in OPA. Under the constant temperature condition of 40 ℃, an amplification curve can be observed from about 10min of reaction, and exponential amplification can be formed within 10-20 min. Has no cross reaction with pathogenic nucleic acids such as PPRV, OSS, BEV, IBRV, BPIV3, MO and the like; the sensitivity is the same as that of the PCR detection method; the stability is good; the exJSRV in lung tissues, nasal fluid and peripheral blood leukocytes of diseased sheep can be effectively detected, and the nucleic acid sequences of the exJSRV and the exJSRV can be accurately distinguished. The fluorescent RPA detection method for the sheep pulmonary adenomatosis virus can achieve the lowest detection limit of 1 ng/mu L, can be used for rapidly diagnosing sheep infected with JSRV on site at the early stage, and has important significance for rapidly identifying and controlling the transmission of OPA.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Fig. 1 shows JSRV identification results, where M: standard molecular weight DL 2000; 1. 2: JSRV diseased lungs; 3: and (5) negative control.
Fig. 2 shows the fluorescent RPA primer screening results, wherein, 1: F1/R1; 2: F2/R2; 3: F3/R3; 4: F4/R4.
Figure 3 shows the optimal reaction temperature screen.
FIG. 4 shows fluorescent RPA-specific detection, wherein 1: JSRV; 2, PPRV; OSS; 4, BEV; IBRV; BPIV 3; MO 7, adding a catalyst; 8, negative control. .
FIG. 5 shows sensitivity assays for fluorescent RPA using diseased lung cDNA as a template, where 1-7: the dilutions of cDNA were 1000 ng/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, respectively; 8: and (5) negative control.
FIG. 6 shows the fluorescent RPA repeatability test using diseased lung cDNA as template, wherein, 1-3: concentration of 100 ng/. mu.L; 4-6: concentration of 10 ng/. mu.L; 7: and (5) negative control.
Fig. 7 shows the results of fluorescent RPA clinical sample detection, wherein 1: positive lung; 2: positive nasal fluid; 3: positive peripheral blood leukocytes; 4: a healthy lung; 5: and (5) negative control.
Fig. 8 shows the PCR clinical sample assay results, wherein 1: positive lung; 2: positive nasal fluid; 3: positive peripheral blood leukocytes; 4: a healthy lung; 5: and (5) negative control.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Primary reagents and instruments
The twist Ampexo kit is a product of twist Dx company; the 8-well isothermal nucleic acid amplification instrument AGS8800 was purchased from Hangzhou reputation technologies, Inc.
Viral sample source and processing
The exJSRV positive lung tissue is stored in the laboratory, and is prepared into cDNA for later use through nucleic acid separation and extraction. Other viral nucleic acids used in this study: peste des Petits Ruminants virus (PPRV), Streptococcus melitensis (OSS) were kept for the laboratory, Bovine Enterovirus (BEV), Infectious Rhinotracheitis virus (IBRV), Bovine Parainfluenza virus type3 (BPIV 3), Mycoplasma pneumoniae (MO) was provided by Probiotics professor Hokkaido university of inner Mongolia.
EXAMPLE 1 preparation and identification of Positive control in a kit
(1) Extraction of total RNA: and extracting RNA from JSRV positive lung tissue by using a total RNA rapid extraction kit.
(2) Reverse transcription: the total RNA extracted above was reverse transcribed into cDNA using methods commonly used in the art.
(3) The PCR primers in Table 1 were used for PCR identification, the target fragment was 1848bp, and the amplification result was consistent with the size of the target fragment (see FIG. 1).
Example 2 design and screening of primers and probes
RPA is a novel isothermal amplification technology, compared with the detection method, the method has the advantages of simple operation, short reaction time (detection result can be observed within 10-20 min), low operation temperature (37-42 ℃) and commercially available freeze-drying reagent, and the method is applied to field diagnosis outside a laboratory. One of the key points of the invention is to design a primer probe based on a recombinase polymerase amplification technology, but the core and difficulty of the RPA method is that the design and the screening of the primer and the probe can not be carried out by auxiliary software and only can depend on manual design.
According to the accession numbers AF105220 and JQ837489 on Genbank, the env gene sequence is downloaded, DNAMAN is used for analyzing the env high-conservation area, and 4 pairs of specific primers and 1 probe are designed. The primers and probes of the present invention were synthesized by Biotechnology engineering (Shanghai) Inc.
TABLE 1 fluorescent RPA composition sequences and PCR primer sequences
Through a large number of comparative experiments, the optimal primer probe for detecting exJSRV is F1/R1 (FIG. 2). Namely:
the forward primer is a nucleotide sequence shown in SEQ ID No. 3;
the reverse primer is a nucleotide sequence shown in SEQ ID No. 4;
the probe is a nucleotide sequence shown as SEQ ID No. 11. The probe was replaced with THF at the 31 st base "a" from the 5 'end, with the T base upstream of the THF site labeling the FAM fluorophore, the T base downstream of the THF site labeling the BHQ1 fluorophore, and the C3 Spacer at the 3' end.
The length of the target fragment obtained by final amplification is 338bp, and the gene sequence is shown as SEQ ID No. 12:
TATGATGTAGTCAGAGTCTTAGGAGAGCAAGTTCAGAGCATTAATTTTCGCATGAAAATCCAATGTCATGCTAACTATAAATGGATTTGTGTTACAAAAAAGCCATACAATACTTCTGATTTTCCATGGGACAAAGTGAAGAAACATTTGCAAGGAATTTGGTTCAATACTAATCTATCGTTAGACCTTTTACAACTGCATAATGAGATTCTTGATATTGAAAATTCGCCGAAGGCTACACTAAATATAGCCGATACTGTTGATAATTTCTTGCAAAATTTATTCTCTAATTTTCCTAGTCTCCATTCGCTGTGGAAAACCCTGATTGGTGTAGGAAT
EXAMPLE 3 fluorescent RPA reaction System and determination of reaction conditions
Fluorescent RPA amplification is carried out by using twist Ampexo Kit, the total reaction volume is 50 muL, wherein, the reduction buffer is 29.5 muL, the template is 1 muL, the forward primer and the reverse primer are respectively 2.1 muL (10 muM), the probe is 0.6 muL (10 muM), DNase/RNase-Free deioned Water is added to supplement 47.5 muL, the mixture is evenly mixed and then is moved into an RPA freeze-dried enzyme powder reaction tube, and then MgAc solution with the concentration of 280mmol/L is added for 2.5 muL. And (3) after uniformly mixing, putting the reaction tube into a constant-temperature nucleic acid amplification instrument, reacting for 20min (taking out the reaction tube at 4min, uniformly mixing, and then putting back into the instrument), and meanwhile, collecting FAM fluorescent signals in the whole reaction process. The reaction temperatures were set to 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃ and 42 ℃ respectively, and the optimal reaction temperature was selected. Amplification curves that observed exponential growth within 20min were judged positive, whereas negative. The optimum reaction temperature was selected to find that the CT value of 11.52 was the lowest at 40 deg.C (FIG. 3), so the optimum reaction temperature for this method was determined to be 40 deg.C.
Example 4 specificity test
And respectively carrying out fluorescent RPA detection by using exJSRV, PPRV, OSS, BEV, IBRV, BPIV3 and MO as templates. The results (FIG. 4) show that only exJSRV had a fluorescence amplification curve and was positive. And the other pathogenic nucleic acids and the negative control have no fluorescence curve and are negative. The established fluorescence RPA method can carry out specificity detection on exJSRV, has no cross reaction with PPRV, OSS, BEV, IBRV, BPIV3 and MO inactivated virus nucleic acid, and has strong specificity.
Example 5 sensitivity test
The diseased lung cDNA is taken as a template, the concentration is 1000 ng/mu L through an enzyme labeling instrument, 10 times of dilution is carried out, and fluorescence RPA detection is carried out. The results (fig. 5) show that: the method can detect the concentration of 1 ng/. mu.L of diseased lung cDNA, and the template of 100 pg/. mu.L, 10 pg/. mu.L and 1 pg/. mu.L and the negative control do not generate obvious amplification curves.
Example 6 repeatability test
Fluorescent RPA replicates were performed at 100 ng/. mu.L and 10 ng/. mu.L concentrations of diseased lung cDNA (FIG. 6). The result shows that the detection results of the method are consistent for the templates with the same concentration, the corresponding fluorescence curves can be observed at the same position, and the stability is good.
Example 7 sample testing
After 1 part of each of lung tissue, nasal fluid and peripheral blood leukocyte of a JSRV infected sheep (from different diseased sheep) is extracted, virus RNA is reversely transcribed into cDNA serving as a template to carry out fluorescence RPA detection. And then selecting healthy sheep lung tissues and carrying out fluorescence RPA detection by the same method. PCR parallel experiments were performed simultaneously, and a negative control was set. The detection results of the samples are as follows (fig. 7 and fig. 8), the detection results of the lung tissues, nasal fluids and peripheral blood leukocytes of the sheep containing the JSRV virus are positive, the detection results of the lung tissues of the healthy sheep and the negative control are negative, and the detection results are consistent with the PCR detection results, which shows that the fluorescence RPA detection method established by the research can effectively detect exJSRV in the diseased sheep.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
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Claims (9)
1. A fluorescent RPA composition for detecting sheep pulmonary adenomatosis virus, which is characterized by comprising the following primers and a probe matched with the primers:
the forward primer is a nucleotide sequence shown as SEQ ID No. 3;
the reverse primer is a nucleotide sequence shown as SEQ ID No. 4;
the probe is a nucleotide sequence shown in SEQ ID No.11, the 31 st base A from the 5 'end of the probe is replaced by THF, the first T base upstream of the THF site is marked with FAM fluorophore, the first T base downstream of the THF site is marked with BHQ1 fluorophore, and the 3' end is marked with C3 Spacer.
2. Use of the fluorescent RPA composition of claim 1 in the preparation of a fluorescent RPA kit for the detection of ovine pulmonary adenomatosis virus.
3. A fluorescent RPA kit for detecting ovine pulmonary adenomatosis virus, said kit comprising the fluorescent RPA composition of claim 1.
4. The fluorescent RPA kit according to claim 3, further comprising a positive control.
5. The fluorescent RPA kit of claim 4, wherein said positive control is in vitro reverse transcribed cDNA of the ovine pulmonary adenomatosis virus env gene.
6. The fluorescent RPA kit of claim 4, further comprising a single-stranded nucleic acid-binding recombinase, a single-stranded DNA-binding protein, a strand-displacing DNA polymerase, a reaction system buffer, and magnesium acetate.
7. A method for detecting ovine pulmonary adenomatosis virus of non-diagnostic interest, said method comprising:
extracting total RNA of a sample to be detected, and performing reverse transcription to obtain cDNA;
formulating a fluorescent RPA reaction system comprising the fluorescent RPA composition of claim 1;
carrying out fluorescent RPA amplification reaction by taking cDNA obtained by reverse transcription as a template;
and (6) judging the result.
8. The detection method according to claim 7, wherein the fluorescent RPA reaction system comprises 2.1. mu.L of 10 μ M forward primer, 2.1. mu.L of 10 μ M reverse primer, and 0.6. mu.L of 10 μ M probe, in a total volume of 50 μ L.
9. The method for detecting according to claim 7, wherein the fluorescent RPA amplification reaction is carried out at a reaction temperature of 40 ℃ and a reaction time of 20 min.
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| CN113373266A (en) | 2021-09-10 |
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