CN116042922A - Tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, kit and application - Google Patents

Abstract

The invention belongs to the technical field of virus molecular detection, and particularly relates to a tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, a kit and application. The nucleotide sequence of the primer related to the technical scheme is as follows: forward primer: 5'-GGGTTTTCACCCTTCCTTTC-3', reverse primer: 5'-TGTTTCWRCDGGGATTACTCCTG-3'; the sequence of the fluorescent probe is as follows: 5'-FAM-TGGGAGACTCCCCAGCCGCGACTCCTT-BHQ1-3'; the kit at least comprises the primer and the fluorescent probe. The fluorescence quantitative PCR detection method for the tomato male plant virus provided by the invention is convenient and quick to operate, can quantitatively detect the tomato male plant virus, has good specificity and high sensitivity, and is suitable for being widely popularized and applied in the field of plant disease diagnosis.

Description

Tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, kit and application

Technical Field

The invention belongs to the technical field of virus molecular detection, and particularly relates to a tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, a kit and application.

Background

The solanaceae plants comprise important vegetables, economic and field crops such as tomatoes (Lycopersicon esclulantum Mill), potatoes (Solanum tuberosum L.), capsicum annuum L.), tobacco (Nicotiana tabacum L.), and the like, are widely cultivated worldwide, and have important economic values. In particular potatoes and tomatoes, which are the first and second most economically important vegetables of the world, are planted in 173 countries worldwide. However, solanaceae plants are also important viral and viroid hosts, and the yield or quality is reduced after infection by the virus or viroid, resulting in serious economic loss. Viroids are a class of single-stranded RNA molecules with closed loops and a molecular weight of about 105Da, containing 246-401 nucleotides. Viroids are one of the smaller pathogens than known viruses that are capable of autonomous replication in host cells, they do not encode proteins, but cause symptoms of varying severity, ranging from minor effects, such as barely visible growth retardation, to deformation, necrosis or chlorosis, and severe dysplasia in the infected plant.

Tomato male strain viroids (Tomato planta macho viroid, TPMVd) are belonging to the family potentilla fusiformica (posiviridae), genus potentilla (posiviridae). The tomato male plant virus is used as an important host, the plant is dwarfed and the yield is reduced after being infected by the tomato male plant virus, and the TPMVd is mechanically transmitted through grafting, budding and cutters and is remotely transmitted through seed and seedling transportation. The amount of imported Solanaceae seeds in China is very large every year, the risk of virus transmission into China is high, and once colonization and diffusion are carried out, serious loss can be caused to the production of Solanaceae crops in China; meanwhile, china is also one of the global main seed reproduction bases of the Solanaceae seeds, and TPMVd can be returned or destroyed when being detected in the export process, so that very serious economic loss is caused.

The virus has not been reported on the Solanaceae plants in China, and researchers in China rarely develop detection research on TPMVd, and at present, related reports such as detection patents and standards specially aiming at the virus are not available in China, which bring about very negative effects on diagnosis and prevention and control of the infection of the TPMVd by the Solanaceae plants, so that a TaqMan real-time fluorescent quantitative RT-PCR detection system of the tomato male strain virus is designed and established, the rapid and accurate detection of the tomato male strain virus is very necessary, and the method has important significance on the aspects of monitoring and early warning of the tomato male strain virus, national gate biosafety and the like.

Disclosure of Invention

The invention aims to provide a tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection primer, a fluorescent probe, a kit and application, and adopts the following specific technical scheme.

A real-time fluorescent quantitative RT-PCR detection fluorescent probe for tomato male strain virus TaqMan has the sequence: 5'-FAM-TGGGAGACTCCCCAGCCGCGACTCCTT-BHQ1-3'.

The nucleotide sequence of the primer used in combination with the fluorescent probe is as follows:

forward primer: 5'-GGGTTTTCACCCTTCCTTTC-3';

reverse primer: 5'-TGTTTCWRCDGGGATTACTCCTG-3'.

A real-time fluorescent quantitative RT-PCR detection kit for tomato male strain virus TaqMan at least comprises the fluorescent probe and the primer.

The application of the TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, primer or kit for detecting the tomato male strain virus is provided.

In particular, tomato male strain viroids are identified from the 6 potato spindle tuber viroids, goldfish latent viroids, capsicum frutescens, tomato acronychia viroids, tomato chlorosis dwarf viroids and tomato male strain viroids.

Moreover, the kit also comprises TaqMan real-time fluorescent PCR amplification premix and positive plasmids.

Furthermore, a positive plasmid was obtained by inserting the sequence shown in SEQ ID No.1 into pGEM-T vector.

The method for detecting tomato male strain viroid by using the kit comprises the following steps:

step 1, extracting RNA of a sample to be detected and performing reverse transcription to obtain cDNA;

step 2, a real-time fluorescence PCR system is as follows: taqMan real-time fluorescence 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 was replenished to a total volume of 25 μl; the real-time fluorescent PCR reaction conditions are as follows: 50 ℃ for 2min, 95 ℃ for 10min, 95 ℃ for 15s, 60 ℃ for 1min,40 cycles;

step 3, judging the detection result: ct value is less than or equal to 35, and a typical amplification curve appears, and the result is positive; no Ct value or amplification curve, and negative result; and (3) when the Ct value is more than 35, the sample is reworked, if the reworked result has no Ct value, the sample is negative, and otherwise, the sample is positive.

Compared with the prior art, the beneficial effect of this technical scheme lies in: 1. the TaqMan real-time fluorescent quantitative RT-PCR detection primer and the fluorescent probe for the tomato male strain virus 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 prove that the primer and the fluorescent probe can accurately identify tomato male plant viroids from 6 potato spindle tuber viroids, the specificity reaches 100 percent, and the 6 potato spindle tuber viroids comprise potato spindle tuber viroids PSTvd, goldfish latent viroids CLVd, capsicum frutescens PCFVd, tomato acronychia viroids TASVd, tomato chlorosis dwarf viroids TCDVd and tomato male plant viroids TPMVd; 3. the kit is simple and convenient to operate, high in specificity and sensitivity, and accurate in quantitative determination within the range of 100 ng/mu L-1 fg/mu L, and the detection limit is 1 fg/mu L; 4. the detection method of the kit is provided, and the quantitative detection of the tomato male strain virus in the test material is realized through a standard curve; the whole process only needs about 1.5 hours, and compared with the conventional PCR technology, the detection time is greatly shortened; the detection result can be directly read by computer software without post-reaction treatment, so that the false positive generation and environmental pollution of the detection result are avoided; 5. has important significance in import and export early warning monitoring of tomato male strain virus, national biological safety and the like.

Drawings

FIG. 1 is a standard curve of a tomato male strain virus (TPMVd) fluorescent quantitative PCR method.

FIG. 2 is an amplification curve of sensitivity analysis of tomato male strain virus (TPMVd) fluorescent quantitative PCR method; and carrying out real-time fluorescence PCR detection analysis on linearity and sensitivity of the 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; 4:100 pg/. Mu.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 a specific assay of tomato male strain virus (TPMVd) fluorescent quantitative PCR method, wherein 1: tomato male strain virus (TPMVd) cDNA;2: mixed cDNAs of other 5 non-target viroids (potato spindle tuber viroids 5 viroids including potato spindle tuber viroids PSTVD, goldfish grass latent viroids CLVd, capsicum fruit viroids PCFVd, tomato acrosis viroids TASVd and tomato chlorosis dwarf viroids TCDVd); 3: mixed cDNA of 6-species viruses (Potato spindle tuber class 6-species viruses including Potato spindle tuber class PSTVD, goldfish latent class CLVd, capsicum fruit class PCFVd, tomato acrosis class TASVd, tomato chlorosis dwarf class TCDVd and tomato male strain class TPMVd) 4: blank control; 5: negative control.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples, which are not intended to limit the scope of the invention.

Example 1

Design and synthesis of tomato male strain viroid fluorescent quantitative PCR primer and probe:

aiming at viroid detection, comparing and analyzing the full-length nucleotide sequence of the potato spindle tuber viroid 6-type virus, and designing a universal primer (PosVdF/-R) based on a nucleotide conservation region:

forward primer Pos-Vd-F:5'-GGGTTTTCACCCTTCCTTTC-3'

Reverse primer Pos-Vd-R:5'-TGTTTCWRCDGGGATTACTCCTG-3'

Selecting a potato spindle tuber viroid 6 kind virus specific region, and designing a specific TaqMan probe aiming at tomato male strain viroid:

TPMVd-Probe:5'-FAM-TGGGAGACTCCCCAGCCGCGACTCCTT-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 the university of martial arts, inc.

Example 2

A real-time fluorescent quantitative RT-PCR detection kit for tomato male strain virus TaqMan at least comprises the primer and a fluorescent probe.

Preferably, in preparation, the detection kit comprises: taqMan real-time fluorescent PCR amplification premix, forward primer, reverse primer, fluorescent probe and positive plasmid.

Wherein, the positive plasmid is tomato male strain virus positive plasmid, which is obtained by inserting a sequence shown in SEQ ID NO.1 into pGEM-T vector. And the vector can proliferate in E.coli DH5 alpha. The plasmid is transformed into E.coli DH5 alpha proliferation, extracted by an alkaline lysis method, purified by a DNA purification kit and stored at-20 ℃.

Taking the kit as an example, the real-time fluorescent quantitative RT-PCR detection method for the tomato male strain virus TaqMan comprises the following steps:

step 1, extracting RNA of a sample to be detected and performing reverse transcription to obtain cDNA;

step 2, a real-time fluorescence PCR system is as follows: taqMan real-time fluorescence 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 was replenished to a total volume of 25 μl; the real-time fluorescent PCR reaction conditions are as follows: 50℃for 2min, 95℃for 10min, 95℃for 15s, 60℃for 1min,40 cycles.

Step 3, judging the detection result

Ct value is less than or equal to 35, and a typical amplification curve appears, and the result is positive. No Ct value or no amplification curve, the result is negative. And (3) when the Ct value is more than 35, the sample is reworked, if the reworked result has no Ct value, the sample is negative, and 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, and only 1.5 hours is needed, and the total time is only 3-4 hours after RNA extraction and reverse transcription preparation are added;

3. the method is easy to implement and simple and convenient to operate.

4. High-flux sample detection can be performed simultaneously;

example 3

Establishment of a quantitative formula and sensitivity test of a kit prepared by using the tomato male strain virus fluorescent quantitative RT-PCR primer.

1) Template preparation:

and (3) carrying out proliferation on a plasmid transformed escherichia coli DH5 alpha containing a tomato male strain viroid gene 360 nucleotide fragment (shown as SEQ ID NO. 1) connected into a pGEM-T vector, extracting by an alkaline lysis method, and purifying by a DNA purification kit to obtain a positive recombinant plasmid standard.

2) Standard curve preparation:

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 real-time example 1 to obtain amplification curves of the standard substances with different concentrations. The amplification curve of this amplification method was obtained with the usual logarithm (log C) of the starting concentration (C) of the standard as the abscissa and the cycle threshold (Ct value) at which fluorescence occurs as the 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.

The PCR amplification reaction was performed using 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, 100 ag/. Mu.L of positive standard plasmid of tomato male strain virus TPMVd as a template, respectively, to obtain a standard curve (FIG. 1) of the fluorescent quantitative PCR method of tomato male strain virus TPMVd, wherein the template concentration in each reaction system has a good linear relationship in the range of 100 ng/. Mu.L to 1 fg/. Mu.L, and the linear relationship between the logarithmic value of the initial template amount (ng/. Mu.L) and the Ct value is

y= -3.8504logx+13.3268, correlation coefficient r2=0.954, according to the expected effect of the experimental design.

FIG. 2 is an amplification curve of sensitivity analysis of tomato male strain virus (TPMVd) fluorescent quantitative PCR method; real-time fluorescent PCR detection assays were performed on linearity and sensitivity of different concentration references, where 1:100 ng/. Mu.L; 2:10 ng/. Mu.L; 3:1 ng/. Mu.L; 4:100 pg/. Mu.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 detection lower limit of the real-time fluorescent quantitative PCR detection method for the tomato male strain virus TPMVd established by the invention is 1 fg/. Mu.L.

3) Fluorescent quantitative RT-PCR detection kit specificity test for tomato male strain virus

The method described in example 1 was used to detect the viral cDNA to be detected in table 1, and a blank control group (double distilled water) and a negative control group (no TPMVd solanaceae plant sample nucleic acid) were simultaneously set, and the viral cDNA to be detected was specifically: 1: tomato male strain virus (TPMVd) cDNA;2: mixed cDNAs of other 5 non-target viroids (potato spindle tuber viroids 5 viroids including potato spindle tuber viroids PSTVD, goldfish grass latent viroids CLVd, capsicum fruit viroids PCFVd, tomato acrosis viroids TASVd and tomato chlorosis dwarf viroids TCDVd); 3: mixed cDNA of 6 kinds of viruses (potato spindle tuber viroid 6 kinds of viruses including potato spindle tuber viroid PSTVD, goldfish latent viroid CLVd, capsicum frutescens viroid PCFVd, tomato acrosis viroid TASVd, tomato chlorosis dwarf viroid TCDVd and tomato male strain viroid TPMVd). The specific amplification curve is shown in FIG. 3.

TABLE 1 tomato male strain virus RT-PCR detection kit specificity test results

Test number	Sample of	Amplification results
			1	TPMVd	+
2	Mixed cDNA of other 5 non-target viroids	-
			3	Mixed cDNA of 6 kinds of viruses	+
4	Blank control	-
			5	Negative control	-

As shown in Table 1, only tomato male strain virus (TPMVd) cDNA and 6 kinds of virus mixed cDNA (potato spindle tuber virus genus 6 kinds of viruses including potato spindle tuber virus PSTVD, goldfish grass latent virus CLVd, capsicum small fruit virus PCFVd, tomato top shrinkage virus TASVd, tomato chlorosis dwarf virus TCDVd and tomato male strain virus TPMVd) can see typical amplification curves, and Ct values are 12.33 and 15.21, which indicates that the tomato chlorosis dwarf virus RT-PCR detection primer has very strong specificity.

Claims (8)

1. A real-time fluorescent quantitative RT-PCR detection fluorescent probe for tomato male strain virus TaqMan is characterized by comprising the following sequences: 5'-FAM-TGGGAGACTCCCCAGCCGCGACTCCTT-BHQ1-3'.

2. The tomato male strain virus TaqMan real-time fluorescence quantitative RT-PCR detection fluorescent probe according to claim 1, wherein the nucleotide sequence of a primer matched with the fluorescent probe is as follows:

forward primer: 5'-GGGTTTTCACCCTTCCTTTC-3';

reverse primer: 5'-TGTTTCWRCDGGGATTACTCCTG-3'.

3. A real-time fluorescent quantitative RT-PCR detection kit for tomato male strain virus TaqMan is characterized in that: comprising at least the fluorescent probe according to claim 1 and the primer according to claim 2.

4. Use of a tomato male strain virus TaqMan real-time fluorescent quantitative RT-PCR detection fluorescent probe, primer or kit according to any one of claims 1-3 for detecting tomato male strain virus.

5. The method of claim 4, wherein the tomato male strain is identified from 6 potato spindle tuber viroids, goldfish latent viroids, capsicum parvovirus, tomato acronychia viroids, tomato chlorosis dwarf viroids and tomato male strain viroids.

6. The kit for real-time fluorescent quantitative RT-PCR detection of tomato male strain viroid TaqMan according to claim 3, wherein the kit is characterized in that: taqMan real-time fluorescent PCR amplification premix and positive plasmid are also included.

7. The tomato male strain virus TaqMan real-time fluorescence quantitative RT-PCR detection kit according to claim 6, wherein the kit is characterized in that: the positive plasmid is obtained by inserting the sequence shown in SEQ ID NO.1 into pGEM-T vector.

8. The kit for detecting the tomato male strain virus TaqMan real-time fluorescence quantitative RT-PCR according to claim 7, wherein the method for detecting the tomato male strain virus by using the kit is as follows:

step 1, extracting RNA of a sample to be detected and performing reverse transcription to obtain cDNA;

step 2, a real-time fluorescence PCR system is as follows: taqMan real-time fluorescence 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 was replenished to a total volume of 25 μl; the real-time fluorescent PCR reaction conditions are as follows: 50 ℃ for 2min, 95 ℃ for 10min, 95 ℃ for 15s, 60 ℃ for 1min,40 cycles;

step 3, judging the detection result: ct value is less than or equal to 35, and a typical amplification curve appears, and the result is positive; no Ct value or amplification curve, and negative result; and (3) when the Ct value is more than 35, the sample is reworked, if the reworked result has no Ct value, the sample is negative, and otherwise, the sample is positive.

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