CN113215323A - Method for detecting BlShV by real-time fluorescent quantitative RT-PCR and kit used by method - Google Patents
Method for detecting BlShV by real-time fluorescent quantitative RT-PCR and kit used by method Download PDFInfo
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Abstract
The invention discloses a method for detecting BlShV by real-time fluorescent quantitative RT-PCR and a kit used by the method. The invention adopts double real-time fluorescent quantitative RT-PCR to detect BlShV, and specifically relates to a rapid detection method for simultaneously detecting 2 target genes by respectively designing primers and probes by taking two target genes of BlShV as targets and optimizing a reaction system and conditions. Compared with the common RT-PCR with a single gene as a target, the detection method has higher accuracy and stronger reliability. The invention provides a more accurate and reliable method for the detection of BlShV, is suitable for detection identification and real-time monitoring of BlShV in agricultural production and entry and exit port quarantine, and has important application value.
Description
Technical Field
The invention belongs to the technical field of plant virus detection, and particularly relates to a method for detecting BlShV by real-time fluorescence quantitative RT-PCR and a kit used by the method.
Background
Blueberries are perennial shrub plants of the genus Vaccinium of the family Ericaceae and originate in North America. The blueberry is called as world third-generation fruit and has high nutritional value and economic value. The virus disease is used as an important disease in blueberry production, and the yield and the quality of the blueberries are seriously influenced. Among the reported Blueberry viruses, the Blueberry shock virus (BlShV) is a virus having a large hazard, and belongs to the brome mosaic virus family (broooviridae), the equiaxed unstable circovirus (ilarivus) genus. BlShV infects almost all blueberry varieties and exhibits similar symptoms. The typical symptom caused by BlShV infecting blueberries is that flowers and leaves are suddenly and completely necrotic during the flowering period, so that no result is obtained, and the loss caused by BlShV infecting blueberries can reach 34-90% according to statistics. The BlShV is mainly spread in a large area by pollen carrying infection by bees in the field, and no effective prevention and treatment agent exists up to now. Therefore, the key of the current control on BlShV is to strengthen the early detection of BlShV and take effective measures in time.
Currently, the major methods for BlShV detection include serological ELISA and molecular biological RT-PCR techniques. Serological ELISA detection has larger limitation, higher requirements on specificity, titer and the like of an antibody, and is easy to generate false positive or false negative results. RT-PCR detection has the advantages of strong specificity, high sensitivity and short detection time, but the reported method has lower accuracy and is probably related to the rapid variation speed of RNA viruses.
Disclosure of Invention
The invention aims to detect BlShV more accurately and reliably.
The invention firstly protects a kit for detecting BlShV, which comprises a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2;
the primer BlShV-f1 is a single-stranded DNA molecule shown in SEQ ID NO. 1;
the primer BlShV-r1 is a single-stranded DNA molecule shown in SEQ ID NO. 2;
the probe BlShV-P1 is a single-stranded DNA molecule shown in SEQ ID NO. 3;
the primer BlShV-f2 is a single-stranded DNA molecule shown in SEQ ID NO. 4;
the primer BlShV-r2 is a single-stranded DNA molecule shown in SEQ ID NO. 5;
the probe BlShV-P2 is a single-stranded DNA molecule shown in SEQ ID NO. 6;
the 5 'ends of the probes BlShV-P1 and BlShV-P2 are both provided with FAM fluorescent labels, and the 3' ends of the probes BlShV-P1 and BlShV-P2 are both provided with MGB modifying groups.
The kit specifically comprises the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2 and the probe BlShV-P2.
In any one of the above-mentioned kits, the molar ratio of the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2 and the probe BlShV-P2 may be 1:1:1:1: 1.
Any one of the above kits may further comprise a random primer, 5 XTR Buffer, RNase inhibitor, reverse transcriptase, dNTPs, 2 XTAQMan PCR Mix, a positive control sample of BlShV, a negative control sample without BlShV and RNase-free ddH2O。
Any one of the above-mentioned kits may specifically comprise the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2, the probe BlShV-P2, a random primer, 5 XTRT Buffer, RNase inhibitor, reverse transcriptase, dNTPs, 2 XTAQMan PCR Mix, a positive control sample of BlShV (as a positive control), a negative control sample without BlShV (as a negative control), and RNase-free ddH2And (C) O.
Any one of the random primers may be specifically a product of Thermo Fisher scientific, inc, catalog number SO 142.
Any one of the above reverse transcriptases is specifically a product of Promega corporation, catalog No. M1701. Any of the 5 × RT Buffer, any of the RNase inhibitor and any of the dNTPs described above are components of a reverse transcriptase.
Any one of the 2 xTaqMan PCR Mix may be a product of Promega corporation, catalog No. A6101.
The preparation method of any one of the above kits also belongs to the protection scope of the invention. The preparation method of any one of the kits comprises the step of separately packaging the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2 and the probe BlShV-P2 in any one of the kits.
The invention also provides application of any one of the kits, which can be at least one of h1) -h 3):
h1) detecting or aiding in the detection of BlShV;
h2) detecting or detecting in an auxiliary way whether the sample to be detected is infected with BlShV;
h3) detecting or detecting in an auxiliary way whether the virus to be detected is BlShV;
the use is for the diagnosis and treatment of non-diseases.
The invention also discloses a method for detecting or assisting in detecting whether a sample to be detected is infected with BlShV, which comprises the following steps:
(a1) respectively obtaining cDNA of a sample to be detected, a positive control sample of BlShV and a negative control sample without the BlShV;
(a2) taking cDNA of a sample to be detected as a template, and performing double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the above kits; taking cDNA of a positive control sample of BlShV as a template, and performing double-fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits to serve as a positive control; taking cDNA of a negative control sample without BlShV as a template, and performing double fluorescence quantification by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kitsQuantitative PCR, as a negative control; with RNase-free ddH2Taking O as a template, and carrying out double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits to serve as a blank control;
(a3) if the Ct value of the sample to be detected is less than or equal to 35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, infecting BlShV by the sample to be detected; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and the Ct value of the sample to be detected is not less than 40, the sample to be detected is not infected with BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and if the Ct value of the sample to be tested is less than 35, retesting is needed;
the method is useful for diagnosis and treatment of non-diseases.
In the above method, the method for obtaining cDNA of a sample to be tested may be: 3 mu L, RNase-free ddH of total RNA of a sample to be detected2Mixing O7 mu L and random primer 1 mu L with concentration of 100 mu mol/L, water bathing at 70 ℃ for 10min, and rapidly ice bathing for 5 min; then 5 mul 5 XRT Buffer, 2 mul dNTPs with the concentration of 10mmol/L, 1 mul RNase inhibitory factor with the concentration of 40U/mul and 1 mul reverse transcriptase with the concentration of 200U/mul are added, water bath at 42 ℃ is carried out for 60min, and finally water bath at 70 ℃ is carried out for 10min, thus obtaining the cDNA of the sample to be detected.
In the above method, the reaction system for performing double fluorescent quantitative PCR is 25 μ L, and comprises 2 μ L of cDNA of a sample to be tested, 10 μmol/L primer BlShV-f 11 μ L, 10 μmol/L primer BlShV-r 11 μ L, 10 μmol/L probe BlShV-P11 μ L, 10 μmol/L primer BlShV-f 21 μ L, 10 μmol/L primer BlV-r 21 μ L, 10 μmol/L probe BlShV-P21 μ L, 2 × TaqMan PCR Mix 12.5 μ L and ddH2O4.5. mu.L.
The invention also discloses a method for detecting or detecting whether the virus to be detected is BlShV in an auxiliary way, which comprises the following steps:
(b1) respectively obtaining cDNA of a virus to be detected, a positive control sample of BlShV and a negative control sample without the BlShV;
(b2) taking cDNA of a virus to be detected as a template, and performing double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits; taking cDNA of a positive control sample of BlShV as a template, and performing double-fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits to serve as a positive control; taking cDNA of a negative control sample without BlShV as a template, and performing double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the above-mentioned kits to serve as a negative control; with RNase-free ddH2Taking O as a template, and carrying out double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits to serve as a blank control;
(b3) if the Ct value of the virus to be detected is less than or equal to 35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, the virus to be detected is BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and the Ct value of the virus to be detected is not less than 40, then the virus to be detected is not BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and if the Ct value of the sample to be tested is less than 35, retesting is needed;
the method is useful for diagnosis and treatment of non-diseases.
In the above method, the method for obtaining the cDNA of the virus to be detected comprises: 3 mu L, RNase-free ddH of total RNA of the virus to be detected2Mixing O7 mu L and random primer 1 mu L with concentration of 100 mu mol/L, water bathing at 70 ℃ for 10min, and rapidly ice bathing for 5 min; then 5. mu.L of 5 XTRT Buffer, 2. mu.L of dNTPs at a concentration of 10mmol/L, 1. mu.L of RNase inhibitor at a concentration of 40U/. mu.L and 1. mu.L of reverse transcriptase at a concentration of 200U/. mu.L, 42 ℃ were addedWater bath is carried out for 60min, and finally water bath is carried out for 10min at 70 ℃, so as to obtain the cDNA of the virus to be detected.
In the above method, the reaction system for performing double fluorescent quantitative PCR is 25 μ L, and comprises 2 μ L of cDNA of the virus to be tested, 10 μmol/L primer BlShV-f 11 μ L, 10 μmol/L primer BlShV-r 11 μ L, 10 μmol/L probe BlShV-P11 μ L, 10 μmol/L primer BlShV-f 21 μ L, 10 μmol/L primer BlV-r 21 μ L, 10 μmol/L probe BlShV-P21 μ L, 2 × TaqMan PCR Mix 12.5 μ L and ddH2O4.5. mu.L.
Any one of the positive control samples of BlShV described above may be blueberry leaves infected with BlShV.
Any of the negative control samples described above that do not contain BlShV can be healthy blueberry leaves that are not infected with BlShV.
Compared with the prior art, the kit and the detection method thereof provided by the invention have the beneficial effects that:
(1) the invention relates to a special double real-time fluorescent quantitative RT-PCR detection method, which comprises 2 sets of specific primers and probes, one set of specific primers and probes is specific to a CP gene of a blueberry shock virus, and the other set of specific primers and probes is specific to an RdRp gene of the blueberry shock virus, so that the invention can simultaneously detect the CP gene and the RdRp gene of the blueberry shock virus at one time. Under the condition that the blueberry shock virus variation is small, the detection results of the 2 sets of primers and the probes are positive; under the condition of large variation of the blueberry shock virus, the detection result of at least 1 set of primers and probes is positive. Compared with molecular detection methods such as RT-PCR and fluorescence RT-PCR which take a single gene as a target, the method has higher accuracy and better reliability.
(2) The invention combines the multigene combination and the real-time fluorescent quantitative RT-PCR detection technology, fully exerts the advantages of strong multigene detection specificity and high real-time fluorescent quantitative RT-PCR detection sensitivity, and obviously improves the specificity and the sensitivity of the detection. On one hand, non-specific amplification is avoided, and detection results of other 6 viruses such as blueberry scorch virus (BlScV), blueberry banding virus (BSSV), blueberry leaf mottle virus (BlMoV), Tobacco Streak Virus (TSV), tobacco ringspot virus (TRSV), tomato ringspot virus (ToRSV) and the like on blueberries are detectedAll fruits are negative; on the other hand, the lowest sensitivity can be detected to dilute to 10-4The detection sensitivity of the RNA of the blueberry shock virus (BlShV) is 10 times higher than that of the common RT-PCR2And (4) doubling.
(3) The PCR detection process is fully automatic, the PCR amplification dynamics can be monitored in real time through software, the detection result can be directly observed on a computer, the complicated steps of electrophoresis, dyeing and the like after the traditional RT-PCR amplification are omitted, the detection time is greatly shortened, and the detection efficiency is improved.
(4) The invention has the advantage of high flux, and can detect a large batch of samples at one time.
The invention provides a method for detecting BlShV by real-time fluorescence quantitative RT-PCR with strong specificity, high sensitivity, simple and convenient operation and intuitive result and a kit used by the method, overcomes the defects and the defects of poor specificity, low accuracy (false positive or false negative result), easy generation of non-specific amplification, easy pollution, complex operation and the like in the prior art, is suitable for detection, identification and real-time monitoring of BlShV in agricultural production and port quarantine of entry and exit, and has important application value.
Drawings
FIG. 1 shows the results of the experiment in step one of example 1; wherein 1 is a combination of CP10 and RdRp10, 2 is a combination of CP1 and RdRp1, 3 is a combination of CP1 and RdRp2, 4 is a combination of CP2 and RdRp3, 5 is a combination of CP2 and RdRp4, 6 is a combination of CP3 and RdRp5, 7 is a combination of CP4 and RdRp5, 8 is a combination of CP5 and RdRp5, 9 is a combination of CP6 and RdRp 493 6, 10 is a combination of CP7 and RdRp7, 11 is a combination of CP8 and RdRp8, and 12 is a combination of CP9 and RdRp 9.
FIG. 2 shows the experimental results of step one in example 2; wherein 1 is a sample to be detected, 2 is a negative control, and 3 is a blank control.
FIG. 3 shows the results of the experiment in step two of example 2; wherein 1 is a sample to be detected, 2 is a negative control, and 3 is a blank control.
FIG. 4 shows the results of the experiment in step three of example 2; wherein 1 is a sample to be detected, 2 is a negative control, and 3 is a blank control.
FIG. 5 shows the result of the specificity test.
FIG. 6 shows the results of the sensitivity detection of BlShV using the kit for detecting BlShV prepared in example 1.
FIG. 7 shows the results of sensitivity detection of BlShV by a conventional RT-PCR method.
FIG. 8 shows the results of the repetitive detection.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Ct value (Cycle threshold) is the number of cycles that the fluorescence signal in the reaction tube has undergone when it reaches a set threshold.
In the following examples, the detection by the general RT-PCR method was carried out as follows:
1) reverse transcription
Adding the total RNA 3 mu L, RNase-free ddH of the sample to be detected into a PCR tube2O7 mu L and primer BlShV-R (sequence: 5'-GACCGGTATCGAACCTTCAC-3') 1 mu L with concentration of 10 mu mol/L, mixing, water bath at 70 ℃ for 10min, ice bath for 5min, sequentially adding 5 XRT Buffer5 mu L, dNTPs 2 mu L with concentration of 10mmol/L, reverse transcriptase 1 mu L with concentration of 200U/mu L and RNase inhibitor 1 mu L with concentration of 40U/mu L, water bath at 42 ℃ for 60min, water bath at 70 ℃ for 10min, cooling to room temperature, and synthesizing cDNA.
2) General RT-PCR reaction
mu.L of the cDNA synthesized in step 1) was added to each PCR tube, and 2 XTaq PCR mix 12.5. mu.L of BlShV-F (sequence: 5'-CGAGTAATCATGGTTTGCAAG-3') 1. mu.L of primer BlShV-R at a concentration of 10. mu. mol/L (sequence: 5'-GACCGGTATCGAACCTTCAC-3') 1. mu.L and ddH2O8.5 mu L, so that the total volume of the reaction is 25 mu L; the mixed reaction solution reacts under the following conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 51 ℃ for 45s, and extension at 72 ℃ for 1min, wherein the total number of cycles is 35, and extension at 72 ℃ is continued for 10min after the last cycle is finished, so that the reaction is finished.
3) Electrophoresis detection of PCR amplification product
Taking 10 mu L of PCR reaction product, detecting by 1.5% agarose gel electrophoresis, observing on a gel imaging system and recording the experimental result. If the PCR amplification products have bright DNA bands at 746bp, the sample to be detected is infected with BlShV, otherwise, the sample to be detected is not infected with BlShV.
In the following examples, the detection by DAS-ELISA method comprises the following steps:
1) coated antibodies
BlShV antibody (product of Agdia) was diluted to working concentration (200-fold) with coating buffer, added to wells of an enzyme-linked plate, 100. mu.L/well, and incubated at 37 ℃ for 2 h.
2) Adding sample
The antibody solution coated in the enzyme linked plate is poured out, and the enzyme linked plate is washed 3 times by PBST buffer solution; add 100. mu.L of sample extract to wells of an ELISA plate at 100. mu.L/well and incubate at 37 ℃ for 2h or 4 ℃ overnight.
3) Enzyme-labeled antibody
An enzyme-labeled antibody (a product of Agdia) was diluted to a working concentration (200 times) with an enzyme-labeled antibody buffer, added to the wells of the ELISA plate, 100. mu.L/well, and incubated at 37 ℃ for 2 hours.
4) Adding substrate
Pouring the enzyme-labeled antibody solution in the enzyme-linked plate, and washing for 3 times by using a PBST buffer solution; the substrate pNPP was prepared to a final concentration of 1mg/mL (as-prepared) with the substrate buffer, added to the wells of the ELISA plate at 100. mu.L/well, and left at room temperature in the dark for about 30-60 min.
5) Reading the absorbance (OD) at 405nm with a microplate reader405nm). If the sample OD405nmValue/negative control OD405nmIf the value is more than or equal to 2, the sample to be detected is infected with BlShV, otherwise, the sample to be detected is not infected with BlShV.
Example 1 preparation of a kit for detecting BlShV
Combination optimization of reagent set for simultaneously detecting CP gene (NCBI Genbank sequence number: KF031042) of BlShV and reagent set for detecting RdRp gene (NCBI Genbank sequence number: KF031041) of BlShV
1. A kit consisting of a primer pair and a fluorescent probe is designed and synthesized according to the nucleotide sequence of the CP gene of BlShV. 10 sets of reagents are synthesized in total, wherein the fluorescent quenching group TAMRA is marked at the 3 ' end of the fluorescent probe of CP1-CP5, the non-fluorescent quenching group is marked at the 3 ' end of the fluorescent probe of CP6-CP10, and the MGB modifying group is connected at the 3 ' end, which is shown in Table 1.
TABLE 1
Note: the primer contains f as the upstream primer and r as the downstream primer.
2. A kit consisting of a primer pair and a fluorescent probe is designed and synthesized according to the nucleotide sequence of the RdRp gene of the BlShV. Synthesizing 10 complete reagents in total, wherein the fluorescent quenching group TAMRA is marked at the 3 ' end of the fluorescent probe of RdRp1-RdRp5, the non-fluorescent quenching group is marked at the 3 ' end of the fluorescent probe of RdRp6-RdRp10, and the MGB modifying group is connected at the 3 ' end, which is shown in Table 2.
TABLE 2
Note: the primer contains f as the upstream primer and r as the downstream primer.
3. Combined optimization of complete set of reagents for simultaneously detecting CP gene of BlShV and complete set of reagents for detecting RdRp gene of BlShV
(1) Reverse transcription
Add Total RNA of BlShV 3. mu. L, RNase-free ddH to PCR tubes2O7 mu L and random primer 1 mu L with concentration of 100 mu mol/L, water bath at 70 ℃ for 10min, and quick ice bath for 5 min; then 5. mu.L of 5 XTT Buffer was added at a concentration of 10mmol/LdNTPs 2 mul, RNase inhibitor 1 mul with concentration of 40U/. mu.L and reverse transcriptase 1 mul with concentration of 200U/. mu.L, water bath at 42 ℃ for 60min, and finally water bath at 70 ℃ for 10min to obtain the cDNA of BlShV.
(2) Double fluorescent quantitative PCR
(1) Preparing a reaction system. The reaction system is 25 mu L, and consists of 2 mu L of cDNA of BlShV, a kit for detecting the CP gene of BlShV, 12.5 mu L of 2 XTaqMan PCR Mix and ddH2And (C) O. The concentrations of the primers and the probes in the kit for detecting the CP gene of BlShV and the kit for detecting the CP gene of BlShV were 10. mu. mol/L, and 1. mu.L of the primers and the probes were added.
(2) Taking the reaction system prepared in the step (1) to perform double fluorescence quantitative PCR.
The reaction procedure is as follows: 2min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
(3) Determination of results
If the Ct value is less than or equal to 35, the combination of the complete set of reagent for detecting the CP gene of the BlShV and the complete set of reagent for detecting the RdRp gene of the BlShV can detect the BlShV; if the Ct value is more than or equal to 40, the combination of the reagent set for detecting the CP gene of the BlShV and the reagent set for detecting the RdRp gene of the BlShV can not detect the BlShV; if the Ct value of 35< the Ct value of the sample to be tested is less than 40, retesting is required.
Some of the results are shown in FIG. 1 and Table 3. The result shows that any one of CP1-CP5 and any one of RdRp1-RdRp5 are combined, the detection effect of each combination on BlShV is not ideal, the Ct value is less than or equal to 35, the Ct value is more than or equal to 40, and the repeatability is poor; any one of CP6-CP10 and any one of RdRp6-RdRp10 are combined, the Ct value of the combination of CP10 and RdRp10 is less than or equal to 35, the result is stable after repeated times, and the Ct value of other combinations is less than or equal to 35, the Ct value is more than or equal to 40, and the repeatability is poor. Thus, a combination of CP10 and RdRp10 was selected for use in preparing a kit for detecting BlShV.
TABLE 3-1
| Name of reagent kit | CP1 | CP2 | CP3 | CP4 | CP5 |
| RdRp1 | - | - | - | - | - |
| RdRp2 | - | - | - | - | - |
| RdRp3 | - | - | - | - | - |
| RdRp4 | - | - | - | - | - |
| RdRp5 | - | - | - | - | - |
Note: negative, + positive.
TABLE 3-2
| Name of reagent kit | CP6 | CP7 | CP8 | CP9 | CP10 |
| RdRp6 | - | - | - | - | - |
| RdRp7 | - | - | - | - | - |
| RdRp8 | - | - | - | - | - |
| RdRp9 | - | - | - | - | - |
| RdRp10 | - | - | - | - | + |
Note: negative, + positive.
Preparation of kit for detecting BlShV
1. A primer pair 1 and a probe BlShV-P1 (i.e., CP10) were designed and synthesized based on the nucleotide sequence of the CP gene of BlShV. Primer pair 1 was constructed from BlShV-f 1: 5 '-CRTCCGATTTGGAGTACGATAAGTT-3' (R is A or G) (SEQ ID NO:1) and BlShV-R1: 5'-TCGGGTAGTCAGACTTAAATTCGA-3' (SEQ ID NO: 2). The probe BlShV-P1 is: 5 '-FAM-TCCGAAGAATACGAATTAGTA-MGB-3' (SEQ ID NO: 3). The 5 'end of the probe BlShV-P1 has a FAM fluorescent label, and the 3' end has a MGB modifying group.
2. Primer pair 2 and probe BlShV-P2 (i.e., RdRp10) were designed and synthesized based on the nucleotide sequence of the RdRp gene of BlShV. Primer pair 2 was constructed from BlShV-f 2: 5'-TGCCTCTAGCGAGTTTTTGCA-3' (SEQ ID NO:4) and BlShV-r 2: 5 '-CCACAAGGTCCGTCACTAATRTGT-3' (R is A or G) (SEQ ID NO: 5). The probe BlShV-P2 is: 5 '-FAM-ATGTGGTTTGGTTCTCAC-MGB-3' (SEQ ID NO: 6). The 5 'end of the probe BlShV-P2 has a FAM fluorescent label, and the 3' end has a MGB modifying group.
3. The kit for detecting the BlShV comprises a primer pair 1, a probe BlShV-P1, a primer pair 2, a probe BlShV-P2, a random primer, 5 xRT Buffer, an RNase inhibitor, reverse transcriptase, dNTPs, 2 xTaqMan PCR Mix, a positive control sample of BlShV, a negative control sample without the BlShV and RNase-free ddH2And (C) O.
Any one of the random primers may be specifically a product of Thermo Fisher scientific, inc, catalog number SO 142.
Any one of the above reverse transcriptases is specifically a product of Promega corporation, catalog No. M1701. Any of the 5 × RT Buffer, any of the RNase inhibitor and any of the dNTPs described above are components of a reverse transcriptase.
Any one of the 2 xTaqMan PCR Mix may be a product of Promega corporation, catalog No. A6101.
Example 2 detection of BlShV Using the BlShV detection kit prepared in example 1
The samples to be tested were blueberry leaves infected with BlShV only.
Firstly, the kit for detecting BlShV prepared in example 1 is adopted to detect BlShV by double fluorescence quantitative PCR
1. Reverse transcription
Adding the total RNA 3 mu L, RNase-free ddH of the sample to be detected into a PCR tube2O7 mu L and random primer 1 mu L with concentration of 100 mu mol/L, water bath at 70 ℃ for 10min, and quick ice bath for 5 min; then 5 XRT Buffer5 μ L, 2 μ L dNTPs with concentration of 10mmol/L, 1 μ L RNase inhibitory factor with concentration of 40U/μ L and 1 μ L reverse transcriptase with concentration of 200U/μ L are added, water bath at 42 ℃ is carried out for 60min, and finally water bath at 70 ℃ is carried out for 10min, thus obtaining the cDNA of the sample to be detected.
2. Double fluorescent quantitative PCR
(1) Preparing a reaction system. Reaction systemThe sample is 25 mu L, and the sample consists of 2 mu L of cDNA of a sample to be tested, 10 mu mol/L BlShV-f 11 mu L, 10 mu mol/L BlShV-r 11 mu L, 10 mu mol/L probe BlShV-P11 mu L, 10 mu mol/L BlShV-f 21 mu L, 10 mu mol/L BlShV-r21 mu L, 10 mu mol/L probe BlShV-P21 mu L, 2 XTaq PCR Mix 12.5 mu L and ddH Manx 12.5 mu L2O4.5. mu.L.
(2) Taking the reaction system prepared in the step (1) to perform double fluorescence quantitative PCR.
The reaction procedure is as follows: 2min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
According to the steps, the total RNA of the sample to be detected is replaced by the RNA of the positive control sample of BlShV, and other steps are not changed and are used as positive controls.
And replacing the total RNA of the sample to be detected with the RNA of the negative control sample without BlShV according to the steps, and taking the RNA as a negative control without changing other steps.
Replacing the total RNA of the sample to be detected with RNase-free ddH according to the steps2O, all other steps were unchanged and used as blank control.
3. Determination of results
If the Ct value of the sample to be detected is less than or equal to 35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, infecting BlShV by the sample to be detected; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and the Ct value of the sample to be detected is not less than 40, the sample to be detected is not infected with BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, if the Ct value of the sample to be tested is 35< 40, retesting is needed.
The results are shown in FIG. 2. The result shows that the typical amplification curve appears only in the blueberry leaves infected with the BlShV, and the Ct value is 21.7.
Secondly, detecting BlShV by single fluorescent quantitative PCR aiming at CP gene
1. Reverse transcription
The same as step 1.
2. Single fluorescent quantitative PCR
(1) Preparing a reaction system. The reaction system is 25 mu L, and consists of 2 mu L of cDNA of a sample to be detected, 10 mu mol/L BlShV-f 11 mu L, 10 mu mol/L BlShV-r 11 mu L, 10 mu mol/L probe BlShV-P11 mu L, 2 XTaqMan PCR Mix 12.5 mu L and ddH2O7.5. mu.L.
(2) Taking the reaction system prepared in the step (1) to perform double fluorescence quantitative PCR.
The reaction procedure is as follows: 2min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
According to the steps, the total RNA of the sample to be detected is replaced by the RNA of the positive control sample of BlShV, and other steps are not changed and are used as positive controls.
And replacing the total RNA of the sample to be detected with the RNA of the negative control sample without BlShV according to the steps, and taking the RNA as a negative control without changing other steps.
Replacing the total RNA of the sample to be detected with RNase-free ddH according to the steps2O, all other steps were unchanged and used as blank control.
3. Determination of results
The same as step one, step 3.
The results are shown in FIG. 3. The result shows that only the blueberry leaves infected with the BlShV show a typical amplification curve, and the Ct value is 23.9.
Thirdly, detecting BlShV by single fluorescent quantitative PCR aiming at RdRp gene
1. Reverse transcription
The same as step 1.
2. Single fluorescent quantitative PCR
(1) Preparing a reaction system. The reaction system is 25 mu L, and consists of 2 mu L of cDNA of a sample to be detected, 10 mu mol/L BlShV-f 21 mu L, 10 mu mol/L BlShV-r21 mu L, 10 mu mol/L probe BlShV-P21 mu L, 2 XTaqMan PCR Mix 12.5 mu L and ddH2O7.5. mu.L.
(2) Taking the reaction system prepared in the step (1) to perform double fluorescence quantitative PCR.
The reaction procedure is as follows: 2min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.
According to the steps, the total RNA of the sample to be detected is replaced by the RNA of the positive control sample of BlShV, and other steps are not changed and are used as positive controls.
And replacing the total RNA of the sample to be detected with the RNA of the negative control sample without BlShV according to the steps, and taking the RNA as a negative control without changing other steps.
Replacing the total RNA of the sample to be detected with RNase-free ddH according to the steps2O, all other steps were unchanged and used as blank control.
3. Determination of results
The same as step one, step 3.
The results are shown in FIG. 4. The result shows that only the blueberry leaves infected with the BlShV show a typical amplification curve, and the Ct value is 26.8.
The results show that the detection results of the 2 sets of primers and the probes are positive under the condition that the blueberry shock virus variation is small; under the condition of large variation of the blueberry shock virus, the detection result of at least 1 set of primers and probes is positive, so that the accuracy of the detection result is fully ensured.
Example 3 specific detection
The samples to be tested were samples infected with BlShV only, blueberry-scorch virus (BlScV) only, blueberry-banding virus (BSSV) only, blueberry-mottle virus (BlMoV) only, tobacco linear virus (TSV) only, tobacco ringspot virus (TRSV) only, or tomato ringspot virus (ToRSV) only.
The sample to be tested was tested according to the method of step one in example 2.
The results of the assay are shown in FIG. 5(1 is a sample infected with BlShV only, 2 is a sample infected with BlScV only, 3 is a sample infected with BSSV only, 4 is a sample infected with BlMoV only, 5 is a sample infected with TSV only, 6 is a sample infected with TRSV only, 7 is a sample infected with ToRSV only, and 8 is a negative control). The results showed that the samples infected with BlShV only exhibited a typical amplification curve with a Ct value of 21.6, whereas the samples infected with BlScV only, BSSV only, BlMoV only, TSV only, TRSV only, ToRSV only, and the negative control exhibited no typical amplification curve.
Therefore, the kit prepared in example 1 has strong specificity for detecting BlShV.
Example 4 sensitivity detection
1. Taking total RNA of BlShV, and using RNase-free ddH2Performing 10-fold gradient dilution on the O to obtain a dilution of 10-1 Dilution 1, dilution degree of 10-2 Dilution 2, dilution degree of 10-3 Dilution 3, dilution degree of 10-4 Dilution 4 of (D) with a dilution of 10-5 Dilution 5, dilution degree of 10-6 Dilution 6 of (2) with a dilution of 10-7The diluent 7 of (1).
2. Total RNA, diluent 1, diluent 2, diluent 3, diluent 4, diluent 5, diluent 6 and diluent 7 of BlShV were each assayed according to the method of step one in example 2.
The results are shown in FIG. 6(1 is total RNA of BlShV, 2 is diluent 1, 3 is diluent 2, 4 is diluent 3, 5 is diluent 4, 6 is diluent 5, 7 is diluent 6, 8 is diluent 7, and 9 is negative control). The results show that the kit prepared in example 1 can detect dilution to diluent 4 at the lowest.
3. And detecting total RNA, diluent 1, diluent 2, diluent 3, diluent 4, diluent 5, diluent 6 and diluent 7 of BlShV by adopting a common RT-PCR method.
The results are shown in FIG. 7(1 is total RNA of BlShV, 2 is diluent 1, 3 is diluent 2, 4 is diluent 3, 5 is diluent 4, 6 is diluent 5, 7 is diluent 6, 8 is diluent 7, and 9 is negative control). The results show that dilution to dilution 2 can be detected at the lowest by the ordinary RT-PCR method.
The results show that the kit prepared in example 1 has high sensitivity when used for detecting BlShV, which is 100 times higher than that of the common RT-PCR method.
Example 5 reproducibility test
Blueberry leaves infected with BlShV only were tested as in step one of example 2.
5 replicates were performed.
The results are shown in FIG. 8 (5 replicates each of 1-5). The result shows that the coefficient of variation of the Ct value is 0.4%, which indicates that the kit prepared in example 1 has good repeatability in detecting BlShV.
Example 6 detection of whether the blueberry sample to be detected is infected with BlShV by using the kit prepared in example 1
The sample to be detected is 120 blueberry samples suspected to be infected with BlShV. Each sample was tested as follows:
1. the method of step one in example 2 was used for detection.
2. The method of step two in example 2 was used for detection.
3. The method of step three in example 2 was used for detection.
4. And detecting by adopting a common RT-PCR method.
5. And detecting by using a DAS-ELISA method.
The results are shown in Table 4: by adopting the method in the first step in the embodiment 2, 15 parts of BlShV infected samples can be detected from 120 parts of samples to be detected, and the detection rate is 12.5%; by adopting the method of the second and third steps in the embodiment 2, 14 samples infected with BlShV can be detected from 120 samples to be detected, the detection rate is 11.7 percent, namely 1 sample in 15 samples detected by the kit is not detected by the real-time fluorescent quantitative RT-PCR method aiming at the CP gene or the RdRp gene; the detection rates of the common RT-PCR method and the DAS-ELISA method are respectively 10.8 percent and 7.5 percent, and the detection rates are obviously lower than that of the kit prepared by the invention.
TABLE 4
| Detection method | Number of samples detected | Percentage of detection (%) |
| Example 2 method of step one | 15 | 12.5 |
| Example 2 Process of step two | 14 | 11.7 |
| Example 2 Process of step three | 14 | 11.7 |
| General RT-PCR | 13 | 10.8 |
| DAS- |
9 | 7.5 |
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> Fujian province agricultural science institute fruit tree institute Fuzhou customs technology center
<120> method for detecting BlShV by real-time fluorescence quantitative RT-PCR and kit used by same
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Claims (10)
1. A kit for detecting BlShV comprises a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2;
the primer BlShV-f1 is a single-stranded DNA molecule shown in SEQ ID NO. 1;
the primer BlShV-r1 is a single-stranded DNA molecule shown in SEQ ID NO. 2;
the probe BlShV-P1 is a single-stranded DNA molecule shown in SEQ ID NO. 3;
the primer BlShV-f2 is a single-stranded DNA molecule shown in SEQ ID NO. 4;
the primer BlShV-r2 is a single-stranded DNA molecule shown in SEQ ID NO. 5;
the probe BlShV-P2 is a single-stranded DNA molecule shown in SEQ ID NO. 6;
the 5 'ends of the probes BlShV-P1 and BlShV-P2 are both provided with FAM fluorescent labels, and the 3' ends of the probes BlShV-P1 and BlShV-P2 are both provided with MGB modifying groups.
2. The kit of claim 1, wherein: the kit consists of the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2 and the probe BlShV-P2.
3. The kit of claim 1, wherein: the kit also comprises a random primer, 5 XTRT Buffer, RNase inhibitor, reverse transcriptase, dNTPs, 2 XTAQMan PCR Mix, a positive control sample of BlShV, a negative control sample without BlShV and RNase-free ddH2O。
4. The kit of claim 3, wherein: the kit comprises the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2, the probe BlShV-P2, a random primer, 5 xRT Buffer, an RNase inhibitor, a reverse transcriptase, dNTPs, 2 xTaqMan PCR Mix, a positive control sample of BlShV, a negative control sample without BlShV and RNase-free ddH2And (C) O.
5. A method for producing a kit according to any one of claims 1 to 4, comprising the step of packaging the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2 and the probe BlShV-P2 in the kit according to claim 1 separately.
6. The use of the kit according to any one of claims 1 to 4, being at least one of h1) -h 3):
h1) detecting or aiding in the detection of BlShV;
h2) detecting or detecting in an auxiliary way whether the sample to be detected is infected with BlShV;
h3) detecting or detecting in an auxiliary way whether the virus to be detected is BlShV;
the use is for the diagnosis and treatment of non-diseases.
7. A method for detecting or assisting in detecting whether a sample to be detected is infected with BlShV comprises the following steps:
(a1) respectively obtaining cDNA of a sample to be detected, a positive control sample of BlShV and a negative control sample without the BlShV;
(a2) taking cDNA of a sample to be detected as a template, and carrying out double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in the kit of claim 1 or 2; double fluorescence quantification using cDNA of a positive control sample of BlShV using the primer BlShV-f1, the primer BlShV-r1, the probe BlShV-P1, the primer BlShV-f2, the primer BlShV-r2 and the probe BlShV-P2 in the kit according to claim 1 or 2PCR, as a positive control; performing double fluorescent quantitative PCR by using cDNA of a negative control sample without BlShV and using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in the kit of claim 1 or 2 to serve as a negative control; with RNase-free ddH2Taking O as a template, and carrying out double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits to serve as a blank control;
(a3) if the Ct value of the sample to be detected is less than or equal to 35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, infecting BlShV by the sample to be detected; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and the Ct value of the sample to be detected is not less than 40, the sample to be detected is not infected with BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and if the Ct value of the sample to be tested is less than 35, retesting is needed;
the method is useful for diagnosis and treatment of non-diseases.
8. A method for detecting or detecting in an auxiliary manner whether a virus to be detected is BlShV or not comprises the following steps:
(b1) respectively obtaining cDNA of a virus to be detected, a positive control sample of BlShV and a negative control sample without the BlShV;
(b2) taking cDNA of a virus to be detected as a template, and carrying out double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in the kit of claim 1 or 2; performing double fluorescence quantitative PCR by using cDNA of a positive control sample of BlShV and using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in the kit of claim 1 or 2 to serve as a positive control; the method of using the primer BlShV-f1, the primer BlShV-r1 and the probe in the kit of claim 1 or 2 as the cDNA of the negative control sample without BlShVPerforming double fluorescent quantitative PCR on BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 to serve as a negative control; with RNase-free ddH2Taking O as a template, and carrying out double fluorescence quantitative PCR by using a primer BlShV-f1, a primer BlShV-r1, a probe BlShV-P1, a primer BlShV-f2, a primer BlShV-r2 and a probe BlShV-P2 in any one of the kits to serve as a blank control;
(b3) if the Ct value of the virus to be detected is less than or equal to 35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, the virus to be detected is BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and the Ct value of the virus to be detected is not less than 40, then the virus to be detected is not BlShV; if the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, and if the Ct value of the sample to be tested is less than 35, retesting is needed;
the method is useful for diagnosis and treatment of non-diseases.
9. The method of claim 7 or 8, wherein:
the method for obtaining the cDNA of the sample to be detected comprises the following steps: 3 mu L, RNase-free ddH of total RNA of a sample to be detected2Mixing O7 mu L and random primer 1 mu L with concentration of 100 mu mol/L, water bathing at 70 ℃ for 10min, and rapidly ice bathing for 5 min; then 5 mul 5 XRT Buffer, 2 mul dNTPs with the concentration of 10mmol/L, 1 mul RNase inhibiting factor with the concentration of 40U/mul and 1 mul reverse transcriptase with the concentration of 200U/mul are added, water bath at 42 ℃ is carried out for 60min, and finally water bath at 70 ℃ is carried out for 10min, thus obtaining the cDNA of the sample to be detected;
the method for obtaining the cDNA of the virus to be detected comprises the following steps: 3 mu L, RNase-free ddH of total RNA of the virus to be detected2Mixing O7 mu L and random primer 1 mu L with concentration of 100 mu mol/L, water bathing at 70 ℃ for 10min, and rapidly ice bathing for 5 min; then 5 mul 5 XRT Buffer, 2 mul dNTPs with the concentration of 10mmol/L, 1 mul RNase inhibitor with the concentration of 40U/mul and 1 mul reverse transcriptase with the concentration of 200U/mul are added, water bath at 42 ℃ is carried out for 60min, and finally water bath at 70 ℃ is carried out for 10min, thus obtaining the cDNA of the virus to be detected.
10. The method of claim 7 or 8, wherein: the reaction system for performing the double fluorescent quantitative PCR is 25 mu L, and comprises 2 mu L of cDNA of a sample to be detected or cDNA of a virus to be detected, 10 mu mol/L primer BlShV-f 11 mu L, 10 mu mol/L primer BlShV-r 11 mu L, 10 mu mol/L probe BlShV-P11 mu L, 10 mu mol/L primer BlShV-f 21 mu L, 10 mu mol/L primer BlShV-r21 mu L, 10 mu mol/L probe BlShV-P21 mu L, 2 XTaqMan PCR Mix 12.5 mu L and ddH2O4.5. mu.L.
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