CN115896355A - Goldfish grass latent viroid TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, kit and application - Google Patents
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Abstract
The invention belongs to the technical field of virus molecule detection, and particularly relates to a fluorescence probe for real-time fluorescent quantitative RT-PCR detection of merremia procumbens latent viroid TaqMan, a kit and application. The nucleotide sequence of the primer related by the technical scheme is as follows: a forward primer: 5'-GGGTTTTCACCCTTCCTTTC-3', reverse primer: 5'-TGTTTCWRCDGGGATTACTCCTG-3'; the sequence of the fluorescent probe is: 5'-FAM-CGTCAGCACCTGCGCTGGTCAAGAGGTT-BHQ1-3'; the kit at least comprises the primer and the fluorescent probe. The fluorescence quantitative PCR detection method for the merremia sibirica latent viroid provided by the invention is convenient and rapid to operate, can be used for quantitatively detecting the merremia sibirica latent viroid, has good specificity and high sensitivity, and is suitable for wide popularization and application in the field of plant disease diagnosis.
Description
Technical Field
The invention belongs to the technical field of virus molecule detection, and particularly relates to a Goldfish occult viroid TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, a kit and application.
Background
Solanaceae plants including tomato (tomato milk), potato (potato L.), chili pepper (Capsicum L.), tobacco (Nicotiana tabacum L.), and other important vegetables, economic and field crops are widely cultivated worldwide and have important economic value. In particular potatoes and tomatoes, which are the first and second most economically important vegetables in the world, are grown in 173 countries throughout the world. However, solanaceae plants are also important virus and viroid hosts, and the yield or quality is reduced after the solanaceae plants are infected by the viruses or the viroids, so that serious economic loss is caused. Viroid is a kind of circular closed single-stranded RNA molecule with molecular weight of about 105Da and containing 246-401 nucleotides. Viroids are among the smaller pathogens that are autonomously replicating in the host cell than the known viruses, they do not encode proteins, but cause symptoms of varying severity, from mild effects, such as virtually invisible growth retardation, to deformation, necrosis or chlorosis, and severe dysplasia in infected plants.
The goldfish grass latent viroid (CLVd) belongs to the genus potato spindle tuber viroid and is one of the most serious diseases on tomatoes. The host range of CLVd is mainly limited to members of the solanaceae family, such as tomato, potato, eggplant, pepper, petunia, and in addition, edible chrysanthemum, cucumber, etc. CLVd propagates primarily mechanically through the seed. The symptoms caused by CLVd are very similar to other potatos fusiform tuber viroids, including developmental delay and the like, and are mainly observed on several solanum plants. In susceptible hosts, symptoms such as distortion and venous necrosis can be found in parts of the plant. Infection of tomato by this virus can lead to smaller fruits and reduced seed yield.
Researchers in China rarely carry out detection research on the snapdragon latent viroid in seeds, and lack corresponding detection technology and detection products, but have the requirement of compulsory detection of the snapdragon latent viroid of tomatoes (S.lycopersicum), wild tomatoes (S.chilense), grams Mei Liusi-based tomatoes (S.chemiewski), solanum floridum (S.peruvianum) and tomatoes (S.pimpinellifolia) abroad.
The virus is not reported on solanaceae plants in China, and related reports such as detection patents and standards specially aiming at the virus are not reported at present, which bring very negative influence on diagnosis and prevention and control of solanaceae plant infected CLVd, so that a TaqMan real-time fluorescent quantitative RT-PCR detection system for the snapdragon latent virus is designed and established, which is very necessary for realizing rapid and accurate detection of the snapdragon latent virus, and has important significance on aspects such as monitoring and early warning of the snapdragon latent virus, national biosafety and the like.
Disclosure of Invention
The invention aims to provide a Goldfish occult viroid TaqMan real-time fluorescent quantitative RT-PCR detection primer, a fluorescent probe, a kit and application.
A TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe for a snapdragon occult viroid has a sequence as follows: 5'-FAM-CGTCAGCACCTGCGCTGGTCAAGAGGTT-BHQ1-3'.
And the nucleotide sequence of the primer matched with the fluorescent probe is as follows:
a forward primer: 5'-GGGTTTTCACCCTTCCTTTC-3';
reverse primer: 5'-TGTTTCWRCDGGGATTACTCCTG-3'.
A Goldfish occult viroid TaqMan real-time fluorescent quantitative RT-PCR detection kit at least comprises the fluorescent probe and the primer.
And the application of the TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, primer or kit for the snapdragon occult viroid in the detection of the snapdragon occult viroid.
In particular, the merremia sibirica latent viroid is identified from 6 potato spiny tuber viroids, namely potato spiny tuber viroid, merremia sibirica latent viroid, capsicum fructicum viroid, tomato topping viroid, tomato chlorotic dwarf viroid and tomato male plant viroid.
Moreover, the kit also comprises TaqMan real-time fluorescence PCR amplification premix and positive plasmid.
Moreover, the positive plasmid is obtained by inserting the sequence shown in SEQ ID NO.1 into a pGEM-T vector.
Moreover, the method for detecting the merremia sibirica latent viroid by using the kit comprises the following steps:
Compared with the prior art, the technical scheme has the beneficial effects that: 1. the provided TaqMan real-time fluorescent quantitative RT-PCR detection primer and fluorescent probe for the merremia sibirica latent viroid are suitable for fluorescent quantitative PCR amplification, have good specificity and high sensitivity, and are suitable for wide popularization and application in the field of plant disease diagnosis; 2. experiments verify that the primer and the fluorescent probe can accurately identify the merremia nivea latent viroid from 6 potato fusiform tuber viroids, the specificity reaches 100 percent, and the 6 potato fusiform tuber viroids comprise potato fusiform tuber viroid PSTVd, merremia nivea latent viroid CLVd, capsicum fructide viroid PCFVd, tomato top-shrinking viroid TASVd, tomato chlorotic dwarf viroid TCDVd and tomato male plant viroid TPMVd; 3. the kit is simple and convenient to operate, strong in specificity and high in sensitivity, the detection limit reaches 1 fg/mu L, and the quantitative determination is accurate within the range of 100 ng/mu L-1 fg/mu L; 4. the detection method of the kit is provided, and realizes the quantitative detection of the merremia sibirica latent viroid in the test material through a standard curve; the whole process only needs about 1.5h, and compared with the conventional PCR technology, the detection time is greatly shortened; the detection result can be directly read by computer software without carrying out reaction post-treatment, so that the generation of false positive of the detection result and the pollution to the environment are avoided; 5. has important significance for the import and export early warning monitoring of the agrimony latent viroid, the national biosafety and other aspects.
Drawings
FIG. 1 is a standard curve of fluorescence quantitative PCR method for Pseudosciaena dactylon latent viroid (CLVd).
FIG. 2 is an amplification curve of sensitivity analysis of the fluorescence quantitative PCR method for the grass carp latent viroid (CLVd); and (3) carrying out real-time fluorescence PCR detection analysis on the linearity and the sensitivity of reference substances with different concentrations, wherein the cDNA content in a reaction system is respectively 1:100 ng/. Mu.L; 2:10 ng/. Mu.L; 3:1 ng/. Mu.L; 4:100pg/μ L;5:10 pg/. Mu.L; 6:1 pg/. Mu.L; 7:100 fg/. Mu.L; 8:10 fg/. Mu.L; 9:1 fg/. Mu.L; 10:100 ag/. Mu.L.
FIG. 3 is an amplification curve of specificity analysis of the fluorescence quantitative PCR method for the grass carp latent viroid (CLVd); wherein, 1: antirrhinum majus latent viroid (CLVd) cDNA;2: mixed cdnas of 5 other non-target viroids (5 viroids of the genus potamoviroid, including potamoviroid PSTVd, pepper kowiid PCFVd, tomato top-dwarf viroid TASVd, tomato chlorotic dwarf viroid TCDVd and tomato male viroid TPMVd); 3:6 kinds of virus mixed cDNA (potato spindle tuber viroid 6 kinds of viruses, including potato spindle tuber viroid 5 kinds of viruses, including potato spindle tuber viroid PSTVd, snapdragon latent viroid CLVd, capsicum fructide viroid PCFVd, tomato top dwarf viroid TASVd, tomato chlorotic dwarf viroid TCDVd and tomato male plant viroid TPMVd); 4: blank control; 5: and (5) negative control.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples, and the present invention is not limited to the examples.
Example 1
Designing and synthesizing a fluorescence quantitative PCR primer and a probe for the merremia sibirica latent viroid:
aiming at viroid detection, the full-length nucleotide sequence of the Betula tuber viroid 6 viroid is compared and analyzed, and a universal primer (PosVdF 1/-R1) is designed based on a nucleotide conserved region:
forward primer Pos-Vd-F1:5'-GGGTTTTCACCCTTCCTTTC-3'
Reverse primer Pos-Vd-R1:5'-TGTTTCWRCDGGGATTACTCCTG-3'
Selecting a specific region of the 6-type viruses of the potato spindle tuber viroid, and designing a specific TaqMan probe aiming at the merremia sibirica latent viroid:
the Probe CLVd-Probe1:5'-FAM-CGTCAGCACCTGCGCTGGTCAAGAGGTT-BHQ1-3', wherein the 5 'end of the Probe is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ1.
Primers and probes were synthesized by Wuhan Tianyihui Biotech, inc.
Example 2
A Goldfish occult viroid TaqMan real-time fluorescent quantitative RT-PCR detection kit at least comprises the primer and a fluorescent probe.
Preferably, the test kit, when prepared, comprises: taqMan real-time fluorescent PCR amplification premix, a forward primer, a reverse primer, a fluorescent probe and positive plasmids.
Wherein the positive plasmid is a snapdragon latent viroid positive plasmid and is obtained by inserting a sequence shown by SEQ ID NO.1 into a pGEM-T vector. And the vector can be propagated in E.coli DH5 alpha. Coli DH5 alpha is transformed by the plasmid, extracted by an alkali cracking method, purified by a DNA purification kit and stored at the temperature of minus 20 ℃.
Taking the kit as an example, the TaqMan real-time fluorescent quantitative RT-PCR detection method for the merremia sibirica latent viroid comprises the following steps:
Ct value is less than or equal to 35, and if a typical amplification curve appears, the result is positive. No Ct value or no amplification curve, the result is negative. And when the Ct value is more than 35, the sample is redone, if no Ct value exists in the redone result, the sample is negative, otherwise, the sample is positive.
The detection method has the advantages that:
1. the sensitivity is high, and the quantitative determination is accurate within the range of 100 ng/mu L-1 fg/mu L;
2. the detection speed is high, only 1 hour, and only 3-4 hours are needed in total by RNA extraction and reverse transcription preparation;
3. the method is easy to implement and simple and convenient to operate;
4. high-flux sample detection can be performed simultaneously.
Example 3
The establishment of a quantitative formula of a kit prepared from the primer of the fluorescence quantitative RT-PCR of the snapdragon latent viroid and a sensitivity test.
1) Preparing a template:
370 nucleotide fragments (shown in SEQ ID NO. 1) containing the snapdragon latent viroid gene are connected into plasmid transformation escherichia coli DH5 alpha of a pGEM-T carrier to be proliferated, then alkaline lysis method extraction is carried out, and the positive recombinant plasmid standard substance is obtained after purification by a DNA purification kit.
2) Preparation of a standard curve:
and (3) carrying out real-time fluorescent quantitative PCR amplification on the standard substances with different concentrations by using the fluorescent quantitative PCR reaction system and the reaction conditions described in the embodiment 1 to obtain amplification curves of the standard substances with different concentrations. The amplification curve of the amplification method is obtained by using the common logarithm (logC) of the initial concentration (C) of the standard as an abscissa and the cycle threshold (Ct value) at which fluorescence occurs as an ordinate. And deducing a linear regression equation of the initial copy number of the standard substance and the cycle threshold value according to the result.
Carrying out PCR amplification reaction by respectively using positive standard quality particles of the snapdragon virus CLVd of 100 ng/mu L, 10 ng/mu L, 1 ng/mu L, 100 pg/mu L, 1 pg/mu L, 100 fg/mu L, 10 fg/mu L, 1 fg/mu L and 100 ag/mu L as templates to obtain a standard curve (figure 1) of the snapdragon virus CLVd fluorescent quantitative PCR method, wherein the graph shows that the template concentration in each reaction system has a good linear relation in the range of 100 ng/mu L to 1 fg/mu L, and the linear relation between the logarithm value of the initial template amount (ng/mu L) and the Ct value is the same as the linear relation
y = -4.3597x +15.0349, correlation coefficient R 2 =0.988, expected effect of experimental design is met.
FIG. 2 is an amplification curve of sensitivity analysis of the fluorescence quantitative PCR method for the grass carp latent viroid (CLVd); and (3) carrying out real-time fluorescence PCR detection analysis on the linearity and the sensitivity of reference substances with different concentrations, wherein 1:100 ng/. Mu.L; 2:10 ng/. Mu.L; 3:1 ng/. Mu.L; 4:100pg/μ L;5:10 pg/. Mu.L; 6:1 pg/. Mu.L; 7:100 fg/. Mu.L; 8:10 fg/. Mu.L; 9:1 fg/. Mu.L; 10:100 ag/. Mu.L. As can be seen from FIG. 2, the lower limit of the detection of the real-time fluorescent quantitative PCR detection method for the merremia procumbens latent viroid CLVd established by the invention is 1 fg/. Mu.L.
3) Specificity test of goldfish occult viroid fluorescent quantitative RT-PCR detection kit
The method described in example 1 is used to detect the cDNA of the virus to be detected in table 1, and a blank control group (double distilled water) and a negative control group (solanaceae plant sample nucleic acid without CLVd) are set at the same time, wherein the cDNA of the virus to be detected specifically comprises: 1: antirrhinum majus latent viroid (CLVd) cDNA;2: mixed cdnas of 5 other non-target viroids (5 viroids of the genus potamoviroid, including potamoviroid PSTVd, pepper kowiid PCFVd, tomato top-dwarf viroid TASVd, tomato chlorotic dwarf viroid TCDVd and tomato male viroid TPMVd); 3:6 kinds of virus mixed cDNA (potato spindle tuber viroid 6 kinds of viruses, including 5 kinds of viruses of potato spindle tuber viroid, including potato spindle tuber viroid PSTVd, snapdragon latent viroid CLVd, capsicum fructide viroid PCFVd, tomato top shrinkage viroid TASVd, tomato chlorotic dwarf viroid TCDVd and tomato plant viroid TPMVd). The specific amplification curve is shown in FIG. 3.
TABLE 1 test results of specificity of RT-PCR detection kit for agrimony latent viroid
| Test number | Sample (I) | |
| 1 | CLVd | + |
| 2 | Mixed cDNAs of 5 other non-target viroids | - |
| 3 | 6 type virus mixed cDNA | + |
| 4 | Blank control | - |
| 5 | Negative control | - |
The test results are shown in table 1, only the goldfish latent viroid (CLVd) cDNA and the 6 kinds of virus mixed cDNA (6 kinds of potato spindle tuber viroid, including 5 kinds of potato spindle tuber viroid, including potato spindle tuber viroid PSTVd, goldfish latent viroid CLVd, pepper fruitlet viroid PCFVd, tomato top-shrinking viroid TASVd, tomato chlorotic dwarf viroid TCDVd and tomato male plant viroid TPMVd) can see typical amplification curves, ct values are 10.16 and 14.06, which indicates that the goldfish latent viroid RT-PCR detection primer has strong specificity.
Claims (8)
1. A TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe for the agrimony latent viroid is characterized in that the sequence is as follows: 5'-FAM-CGTCAGCACCTGCGCTGGTCAAGAGGTT-BHQ1-3'.
2. The TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe for the snapdragon occult viroid according to claim 1, wherein the nucleotide sequence of a primer used in cooperation with the fluorescent probe is as follows:
a forward primer: 5'-GGGTTTTCACCCTTCCTTTC-3';
reverse primer: 5'-TGTTTCWRCDGGGATTACTCCTG-3'.
3. A real-time fluorescent quantitative RT-PCR detection kit for a merremia sibirica latent viroid TaqMan is characterized in that: comprising at least the fluorescent probe of claim 1 and the primer of claim 2.
4. The application of the Goldfish grass latent viroid TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, primer or kit in the detection of the Goldfish grass latent viroid according to any one of claims 1 to 3.
5. The use according to claim 4, wherein the pseudolaric latent virus is identified from the 6 genus potatoses tuberoid virus, goldfish latent virus, capsicum fructide virus, tomato topdown virus, tomato chlorotic dwarf virus and tomato male plant virus.
6. The TaqMan real-time fluorescent quantitative RT-PCR detection kit for the agrimonia procumbens latent viroid according to claim 3, wherein: also comprises TaqMan real-time fluorescence PCR amplification premix and positive plasmid.
7. The TaqMan real-time fluorescent quantitative RT-PCR detection kit for the agrimonia procumbens latent viroid according to claim 6, wherein: the positive plasmid is obtained by inserting the sequence shown in SEQ ID NO.1 into pGEM-T vector.
8. The TaqMan real-time fluorescent quantitative RT-PCR detection kit for the snapdragon occult viroid according to claim 7, wherein the method for detecting the snapdragon occult viroid by using the kit comprises the following steps:
step 1, extracting RNA of a sample to be detected and carrying out reverse transcription to obtain cDNA;
step 2, the real-time fluorescent PCR system is as follows: taqMan real-time fluorescent PCR amplification premix solution 12.5. Mu.L, upstream and downstream primers (10. Mu. Mol/mL) each 1.0. Mu.L, taqMan probe (10. Mu. Mol/mL) 0.5. Mu.L, cDNA template 3.0. Mu.L, and ddH 2 O supplementFilling the mixture to a total volume of 25 mu L; the real-time fluorescent PCR reaction conditions are as follows: 50 ℃ for 2min, 95 ℃ for 10min, 95 ℃ for 15s, 60 ℃ for 1min;
and 3, judging a detection result: ct value is less than or equal to 35, and if a typical amplification curve appears, the result is positive; no Ct value or no amplification curve, and negative result; and when the Ct value is more than 35, the sample is redone, if no Ct value exists in the redone result, the sample is negative, otherwise, the sample is positive.
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Citations (2)
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| CN103667527A (en) * | 2013-12-03 | 2014-03-26 | 黑龙江出入境检验检疫局检验检疫技术中心 | Liquid phase chip detection primer of potato spindle tuber viroid, and detection method thereof |
| CN113774053A (en) * | 2021-08-19 | 2021-12-10 | 中国农业科学院植物保护研究所 | Nucleic acid probe and kit for simultaneously detecting multiple tomato viruses and application of nucleic acid probe and kit |
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