CN110894536A - Qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma - Google Patents
Qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma Download PDFInfo
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Abstract
The invention belongs to the technical field of plant quarantine, and relates to a qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma. According to the difference of the apricot chlorotic leafroll phytoplasma and other phytoplasmas on a 16S rDNA gene sequence, a primer pair ACLR-F1/ACLR-R1 and a Probe ACLR-Probe for specifically detecting the apricot chlorotic leafroll phytoplasma are designed, the Ct value is determined by the established real-time fluorescence quantitative PCR detection method of the apricot chlorotic leafroll phytoplasma, and the quality of the apricot chlorotic leafroll phytoplasma can be determined according to the positive judgment standard of the real-time fluorescence quantitative PCR detection result; and calculating the copy concentration of the 16S rDNA gene fragment of the apricot chlorotic leafroll phytoplasma in the detected sample by a pre-established fluorescence quantitative PCR standard curve equation, and accordingly obtaining the number of the apricot chlorotic leafroll phytoplasma in the sample, thereby realizing quantitative detection. The invention has the advantages of high sensitivity, strong specificity, good repeatability, high flux and the like, and can be widely applied to the fields of plant quarantine, plant protection, scientific research and the like.
Description
Technical Field
The invention belongs to the technical field of plant quarantine, relates to a quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma, and particularly relates to primers, probes and a method for qualitatively and quantitatively detecting the Xinjiang isolate of apricot chlorotic leafroll phytoplasma by using a real-time fluorescent quantitative PCR (polymerase chain reaction) technology.
Background
Apricot Chlorosis Leaf Roll (ACLR) is a highly lethal phytoplasma disease that damages the apricot tree. China, Europe and North America all list the pest as entry plant quarantine pest. The disease is firstly discovered on French apricot trees in 1973, and then is sequentially discovered on other stone fruit tree species in Europe such as amygdalus mongholicus, European plums, cherries, peaches, nectarines and other fruit trees, so that the disease becomes a main disease influencing the commercial cultivation of the stone fruit trees in Europe. Although symptoms of apricot chlorosis leaf roll disease reported in different countries are similar, pathogens of apricot chlorosis leaf roll disease reported in each country include multiple phytoplasmas with different classification positions. ACLR pathogens such as turkish, belgium, czech, etc. were identified as members of the apple cluster group (16 SrX). The ACLR pathogen, occurring in libanus and germany, was identified as a member of the flaviviation group of emerald chrysanthemum (16 Sr i). In 2007, Li Wen Hui et al found a suspected disease strain of apricot chlorosis leaf curl in Xinjiang apricot orchard for the first time, the symptoms mainly show that leaves turn up along the main pulse to be rolled into a cylinder shape, the early senescence and fruit dropping result, the fruit is small and bad in taste, the yield of light people is reduced, the whole tree body of heavy people dies, and the pathogen causing Xinjiang apricot chlorosis leaf curl is determined to be phytoplasma through electron microscope observation. The sequence determination and analysis of the phytoplasma 16SrDNA gene in Xinjiang apricot chlorotic leaf roll disease strains in 2010, Hanbing and the like find that the Xinjiang isolates of apricot chlorotic leaf roll phytoplasma belong to members of the elm etiolate group (16 Sr V).
Apricot chlorosis leaf roll disease is wide in distribution range, high in transmission speed and strong in pathogenicity, once a tree body is infected with a disease, the apricot chlorosis leaf roll disease is carried in life, and the apricot chlorosis leaf roll disease is difficult to cure through a traditional disease control technology and a traditional cultivation technology. The method is to fundamentally solve the problem of prevention and control of apricot chlorosis leaf curl disease by adopting effective preventive measures, strengthen quarantine inspection of seedlings (scions and stocks), establish a disease-free seedling breeding system by adopting healthy seedlings (stocks and scions), fundamentally stop pathogeny from the source and be the fundamental way of controlling the occurrence and spread of apricot chlorosis leaf curl disease, and establish and apply an advanced pathogeny detection technology. In recent years, as the molecular biology technology is introduced into the research field of phytoplasma diseases, the phytoplasma identification and detection technology is developed rapidly, such as common PCR, nested PCR and other techniques, greatly improves the detection sensitivity and the identification level of phytoplasma, but the methods have complex whole process and many operation steps, and requires PCR post-treatment such as agarose gel electrophoresis and ethidium bromide staining, ultraviolet light observation or detection by polyacrylamide gel electrophoresis and silver staining, if species or groups are to be identified, restriction enzyme polymorphism analysis (RFLP) is also performed, or the nucleic acid sequence determination and homology comparison need various instruments, and are time-consuming and labor-consuming, the used staining agent ethidium bromide is harmful to human bodies, these complicated experimental procedures provide opportunities for contamination and false positives, which severely affect the correct determination of the results. Therefore, a simple, rapid, sensitive and accurate real-time fluorescence quantitative PCR detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma is developed to meet the requirement of quarantine work.
The fluorescent PCR (FQ-PCR) technology is a new nucleic acid quantitative technology developed by PerkinElmer company 1995 in America, and the method is continuously developed and perfected since the generation, wherein the TaqMan probe real-time fluorescent quantitative PCR technology is that a pair of primers is added in a PCR reaction system, a specific TaqMan probe marked with 2 fluorescent groups is added at the same time, the TaqMan probe is hybridized with a PCR product in the PCR amplification process to cause a reporter group to emit a fluorescent signal, and the initial template is qualitatively and quantitatively analyzed by the real-time detection of the fluorescent signal of each cycle product in the PCR amplification reaction. Compared with the conventional PCR detection method, the method has the advantages of strong specificity, short time consumption, high sensitivity, good stability, quantification and the like.
At present, real-time fluorescent quantitative PCR detection methods for detecting apricot chlorotic leafroll phytoplasma are established at home and abroad, but the methods are primers and probes designed according to conserved genes of the apple cluster group (16 SrX) phytoplasma, so the methods are not suitable for qualitative and quantitative detection of Xinjiang isolates of apricot chlorotic leafroll phytoplasma which belong to the elm etiolate group (16 Sr V) in classification. In addition, although a real-time fluorescence PCR detection method for the members of the elm etiolation group (16 Sr V) of the phytoplasma is established at home and abroad, the method can only realize the specificity detection among the phytoplasma groups and cannot distinguish and identify the phytoplasma in the elm etiolation group of the phytoplasma. On the basis of analyzing and comparing a large amount of genetic information of the phytoplasma, the invention establishes the real-time fluorescent quantitative PCR detection method for the specificity of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma, can better distinguish the phytoplasma among different groups and in the groups, and can greatly improve the detection efficiency of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma, thereby meeting the requirements of relevant quarantine work in China.
Disclosure of Invention
The invention aims to provide a qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma, which has the function of quickly, qualitatively and quantitatively detecting the Xinjiang isolate of apricot chlorotic leafroll phytoplasma. The invention has simple operation, easy mastering and high detection precision, and can be widely applied to daily detection work of laboratories and the like. The technical scheme adopted by the invention for achieving the purpose is as follows.
(1) Primers and probes for real-time fluorescent quantitative PCR detection of Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are designed and provided. The sequences of the primers were: an upstream primer ACLR-F1: 5'-CCCTGGTAGTCCACGCTGTAG-3'; the downstream primer ACLR-R1: 5'-AGACCTGGTAAGGTTTTTCGTGTAC-3'. The sequence of the probe is: ACLR-Probe 5 '-FAM-TTAATGTGTTAACTTCAGTACCG-TAMRA-3'.
(2) The method comprises the steps of firstly preparing a reaction template from a sample to be detected for the apricot chlorotic leafroll phytoplasma Xinjiang isolate, establishing and optimizing a real-time fluorescent quantitative PCR reaction system, and then amplifying and collecting fluorescence of the reaction template by a fluorescent quantitative PCR instrument by setting a real-time fluorescent PCR reaction program.
(3) And finally, the Ct value of each sample is given according to the instrument, and the detection result is analyzed.
Specifically, the invention provides a qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma, which comprises the following steps.
(1) As the core of the invention, a group of highly sensitive and specific primers and probes are provided for the real-time fluorescent quantitative PCR detection of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma, and the detection sensitivity of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma is further improved.
The sequences of the primers were: an upstream primer ACLR-F1: 5'-CCCTGGTAGTCCACGCTGTAG-3'; the downstream primer ACLR-R1: 5'-AGACCTGGTAAGGTTTTTCGTGTAC-3'. The sequence of the probe is: ACLR-Probe: 5'-TTAATGTGTTAACTTCAGTACCG-3'.
The design and synthesis method of the primer and the probe for real-time fluorescent quantitative PCR detection of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma comprises the steps of firstly collecting 16S rDNA nucleic acid sequences of all the phytoplasma and representative bacteria in NCBI, comparing and analyzing the sequences by using Omiga software, finding out gene difference sites of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma and other phytoplasma, screening out a pair of primers by using software Beacon Designer 7.0, setting a fluorescent Taqman probe in an amplification region of the primer pair, marking a report fluorescent dye at the 5 'end of the probe, and marking a quenching fluorescent dye at the 3' end of the probe. After the PCR primer pair and the probe of the specificity of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are designed, the sequence specificity is analyzed by adopting online homology comparison, and the result shows that the primer and the probe design region of the specificity of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are specific to the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma, species which can be matched with the primer pair and cause amplification except the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are not found, and the specificity of an amplification product can be primarily ensured. TaqMan probe and primer pairs were synthesized by Biotech.
The size of the amplified fragment of the real-time fluorescent PCR primer pair is 190 bp, the 5 'end of the specific fluorescent probe of the invention is provided with a report fluorescent dye FAM label, and the 3' end is provided with a quenching fluorescent dye TAMRA label.
(2) Extraction of total plant DNA: extracting total DNA of apricot branch phloem or vein by using Plant Genomic extraction Kit (Plant Genomic DNA Kit, TIANGEN), and storing in a refrigerator at-40 deg.C for use.
(3) The extracted total plant DNA is taken as a template, a positive control, a healthy plant total DNA control and a sterile double distilled water control are respectively arranged, and the designed primer and probe are adopted to carry out real-time fluorescence quantitative PCR detection.
The reaction system for real-time fluorescent quantitative PCR detection is 25 muL: comprises 1.0 muL of DNA template, 10 XPCR buffer2.5 muL, 25 mm/muL MgCl23.2 µL，2.5 mmol/L dNTPs 2.0 µL，2.5 U/µL ExTaqUpstream primer ACLR-F10.6 muL of DNA polymerase 0.5 muL, 10 mumol/L, downstream primer ACLR-R10.6 muL of 10 mumol/L, TaqMan Probe ACLR-Probe 0.8 muL of 10 mumol/L, Bovine Serum Albumin (BSA) 5.0 muL of 1.0 mg/mL, complement sterilization ddH2O to 25 μ L.
Aiming at the primer, the probe and the PCR reaction system, the PCR reaction program is set as that the pre-denaturation is carried out for 5 min at the temperature of 95 ℃; denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 20 s for 40 cycles, and fluorescence collection at 72 ℃ by setting FAM fluorescence channel.
(4) After the reaction is finished, recording the Ct value of the PCR amplification curve of each detection sample, and judging whether the Xinjiang separator of the apricot chlorotic leafroll mycoplasma exists in the sample to be detected according to the Ct value of the reaction of each sample and the positive judgment standard of the real-time fluorescent quantitative PCR detection result of the Xinjiang separator of the apricot chlorotic leafroll mycoplasma, so that the qualitative detection of the Xinjiang separator of the apricot chlorotic leafroll mycoplasma can be realized; and calculating the copy concentration of the Xinjiang isolate 16S rDNA gene fragment of the apricot chlorotic leafroll phytoplasma in the detected sample through a pre-established standard curve equation of fluorescent quantitative PCR reaction, thereby obtaining the content of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma in the sample and further realizing quantitative detection.
(5) The positive judgment standard of the real-time fluorescent quantitative PCR detection result of the Xinjiang isolate of the apricot chlorotic leafroll mycoplasma is as follows: if the Ct value of the sample fluorescence PCR amplification curve is less than or equal to 34 and the negative control and the blank control have no amplification curve, judging that the sample to be detected contains the Xinjiang separator of the apricot chlorotic leafroll phytoplasma, otherwise, judging that the sample does not contain the Xinjiang separator of the apricot chlorotic leafroll phytoplasma; the standard curve equation of the fluorescent quantitative PCR reaction is as follows: y = -3.476x +40.882, wherein x is the logarithm of the copy number of Xinjiang isolate 16S rDNA gene fragment of apricot chlorotic leafroll mycoplasma in the tested sample, and y is a Ct value.
By implementing the specific invention content of the invention, the following effects can be achieved:
the invention fully considers the particularity of quarantine work, develops a set of real-time fluorescent quantitative PCR probes and primers aiming at the characteristics of Xinjiang isolate of apricot chlorotic leafroll phytoplasma, can specifically detect the Xinjiang isolate of apricot chlorotic leafroll phytoplasma, and excludes other phytoplasma, and has the detection sensitivity of 28 copies/mL. In addition, the method for detecting the Xinjiang isolate content of the apricot chlorotic leafroll phytoplasma in the sample can be rapidly and accurately detected by adopting the real-time fluorescent quantitative PCR primer and the probe, and provides a new means and basis for detecting the bacteria of the nursery stock and the scion and identifying the apricot chlorotic leafroll disease resistant variety.
Compared with the prior art, the invention has the beneficial effects that:
(1) the sensitivity is high: the sensitivity of detecting Xinjiang isolate of apricot chlorotic leafroll phytoplasma is more than 100 times higher than that of the common PCR method.
(2) The specificity is strong: the invention only designs specific primers and probes for Xinjiang isolate of apricot chlorosis leaf roll phytoplasma, and selects Xinjiang isolate of apricot chlorosis leaf roll phytoplasma (elm yellowing group), grape golden yellow phytoplasma (elm yellowing group), jujube witches phytoplasma (elm yellowing group), alfalfa arbuscular phytoplasma (elm yellowing group), paulownia fortunei arbuscular phytoplasma (green chrysanthemum yellowing group), willow arbuscular phytoplasma (green chrysanthemum yellowing group) and healthy apricot tree as objects to carry out specificity detection, and the result shows that the detection accuracy is high and the specificity is strong.
(3) The detection time is short: compared with the conventional PCR detection method, the method saves time.
(4) And (3) pollution reduction: because the amplification is done in a closed system and the real-time measurements taken, the possibility of contamination is greatly reduced and no post-amplification manipulations are necessary.
(5) Can be used for qualitative analysis and quantitative analysis.
(6) Safety to humans and the environment: toxic reagents such as ethidium bromide and the like are not used in the detection process, and the method is very safe to people and environment.
In a word, the method is simple and convenient to operate, rapid, efficient and high in flux, and has high sensitivity, repeatability and specificity.
Drawings
FIG. 1 shows a standard curve of real-time fluorescence PCR detection method for Xinjiang isolate of apricot chlorotic leafroll Phytoplasma.
FIG. 2 shows the results of the sensitivity test of the Xinjiang isolate real-time fluorescent PCR detection method for the apricot chlorotic leafroll phytoplasma. In the figure, the concentration of the plasmid template to be adopted is 2.8 multiplied by 10 for the curves 1 to 9 respectively8Amplification at 2.8 copies/mL, curve 10 is a negative control.
FIG. 3 shows the results of the sensitivity tests of the conventional PCR detection method for Xinjiang isolate of the apricot chlorotic leafroll Phytoplasma. In the figure, the M channel indicates 100bp DNA Ladder, and the concentration of the plasmid template to be adopted in lanes 1-9 is 2.8 × 108Amplification at 2.8 copies/mL, lane 10 is a negative control.
FIG. 4 shows the specific test results of real-time fluorescent PCR detection method of Xinjiang isolate of Phytoplasma pruriens of apricot chlorotic leafroll. In the figure, curves 1-6 respectively represent the results of the fluorescent quantitative PCR detection by adopting healthy apricot trees, grape golden yellow phytoplasma, jujube witches broom phytoplasma, alfalfa arbuscular phytoplasma, paulownia arbuscular phytoplasma and willow arbuscular phytoplasma, and curve 7 represents the result of the fluorescent quantitative PCR detection by adopting apricot chlorotic leafroll phytoplasma.
Detailed Description
The invention will be further illustrated with reference to the following specific examples.
It will be understood by those skilled in the art that these examples are only for illustrating the present invention and do not in any way limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Test drugs:plant Genomic extraction Kit (Plant Genomic DNA Kit, TIANGEN), pMDTM19T, deoxynucleotide triphosphates (dNTPs), a plasmid mini-extraction Kit, Bovine Serum Albumin (BSA), a common agarose gel DNA recovery Kit (TIANgel Midi Purification Kit) and RealMasterMix (Probe) were purchased from Beijing Tiangen Biochemical technology, Inc.
The instrument equipment comprises: real-time fluorescent PCR instrument (Roche Lightcycle 2.0), ND-1000 nucleic acid/protein detector (Nano Drop company).
All reagents and equipment selected for use in the present invention are well known in the art and are not intended to limit the practice of the present invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the present invention.
EXAMPLE 1 preparation of primers and probes
1. Design and Synthesis of primers and probes
The design and synthesis method of the primer and the probe for real-time fluorescent quantitative PCR detection of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma comprises the steps of firstly collecting 16S rDNA nucleic acid sequences of all the phytoplasma and representative bacteria in NCBI, comparing and analyzing the sequence by using Omiga software, finding out gene difference sites of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma and other phytoplasma, screening out a pair of primers by using software Beacon Designer 7.0, setting a fluorescent Taqman probe in an amplification region of the primer pair, marking a report fluorescent dye at the 5 'end of the probe, and marking a quenching fluorescent dye at the 3' end of the probe. After the PCR primer and the probe for the specificity of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are designed, the sequence specificity is analyzed by adopting online homology comparison, and the result shows that the primer and the probe for the specificity of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are specific to the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma, and species which can be matched with the pair of primers and cause amplification except the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma are not found, so that the specificity of an amplification product can be ensured preliminarily. The primers and probes were synthesized by Biotechnology Limited of Beijing ancient China.
The sequences of the primers were: an upstream primer ACLR-F1: 5'-CCCTGGTAGTCCACGCTGTAG-3'; the downstream primer ACLR-R1: 5'-AGACCTGGTAAGGTTTTTCGTGTAC-3'. The sequence of the probe is: ACLR-Probe: 5'-TTAATGTGTTAACTTCAGTACCG-3'.
The size of the amplified fragment of the real-time fluorescent PCR primer pair is 190 bp, the 5 'end of the specific fluorescent probe of the invention is provided with a report fluorescent dye FAM label, and the 3' end is provided with a quenching fluorescent dye TAMRA label.
2. Preparation of primers and probes
After the primers and the probes are synthesized, the primers and the probes are respectively diluted to 10 mu mol/L by using sterilized double distilled water, and the primers and the probes are placed in a refrigerator at the temperature of-40 ℃ and are kept in a dark place for later use.
Example 2 establishment of real-time fluorescent PCR detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma
1. Extraction of total plant DNA: extracting total DNA of apricot branch phloem or vein by using Plant Genomic extraction Kit (Plant Genomic DNA Kit, TIANGEN), and storing in a refrigerator at-40 deg.C for use.
2. PCR amplification of Xinjiang isolate 16S rDNA of apricot chlorotic leafroll mycoplasma
The common primer pair R16mF2: 5'-CATGCAAGTCGAACGGA-3' and R16mR2: 5'-CTTAACCCCAATCATCGA-3' of the phytoplasma 16S rDNA gene of the apricot chlorotic leafroll are used for amplifying the gene segment of the phytoplasma 16S rDNA. PCR reaction system (25 μ L): 2.0 muL of DNA template, 1.0 muL of primers (10 mumol/L), 1.0 muL of dNTP (10mmol/L), and 10 XPCR Buffer (2.5mmol/L MgCl)2) 2.5µL，TaqDNA polymerase (2.5U/. mu.L) 0.3. mu.L, complement ddH2O to 25.0 muL. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, renaturation at 50 ℃ for 45s, extension at 72 ℃ for 1min, 35 cycles; extension at 72 ℃ for 10 min.
3. Construction of Positive plasmids
Cutting the specific band in the PCR amplification product, and recovering and purifying by using an agarose gel recovery kit. Recovered PCR product and vector pMDTM19T at 16 ℃ for 2h, transforming the ligation products into DH5 α competent cells, and culturing the cells on LB agar plate medium containing X-Gal (20 g/L), IPTG (50 g/L), Amp (100 mg/L) at 37 ℃ for 14-16 hForming a single colony. Inoculating the screened positive single colony in LB liquid culture medium containing Amp (100 mg/L), culturing overnight at 37 ℃, extracting plasmid, performing PCR amplification and enzyme digestion identification. The target gene was confirmed to be correctly cloned into the vector by sequencing and identification by Shanghai's institute of technology, and the plasmid concentration was measured by a nucleic acid protein analyzer (NanoDroP, ND-1000) and converted into copy number, and the plasmid was stored at-20 ℃ for use and diluted 10-fold in gradient before use.
4. Establishment and optimization of apricot chlorosis leaf curl disease real-time fluorescence PCR reaction system and conditions
Establishing a real-time fluorescent quantitative PCR detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma by using the constructed positive plasmid containing the target gene as a DNA template, optimizing a PCR reaction system and reaction conditions, and determining that the reaction system for real-time fluorescent quantitative PCR detection is 25 mu L: comprises 1.0 muL of DNA template, 2.5 muL of 10 XPCR Buffer, 25 mm/uL MgCl23.2 µL，2.5 mmol/L dNTPs 2.0 µL，2.5 U/µL ExTaqUpstream primer ACLR-F10.6 muL of DNA polymerase 0.5 muL, 10 mumol/L, downstream primer ACLR-R10.6 muL of 10 mumol/L, TaqMan Probe ACLR-Probe 0.8 muL of 10 mumol/L, Bovine Serum Albumin (BSA) 5.0 muL of 1.0 mg/mL, complement sterilization ddH2O to 25 μ L. The fluorescent quantitative PCR reaction program is that the pre-denaturation is carried out for 5 min at the temperature of 95 ℃; denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 20 s for 40 cycles, and fluorescence collection at 72 ℃ by setting FAM fluorescence channel.
5. Establishment of Standard Curve and determination of sensitivity
Dilution with 10-fold gradient (2.8X 10)82.8 × 10 copies/mL) plasmid DNA as a template, detecting on a fluorescence PCR instrument to obtain an amplification kinetic curve and a corresponding standard curve, and evaluating the sensitivity according to the detected lowest copy number. Meanwhile, PCR sensitivity determination is carried out by using the plasmid DNA which is subjected to gradient dilution by using the phytoplasma 16S rDNA gene universal primer pair R16mF2/R16mR2 as a template, and the sensitivity is compared with the fluorescence quantitative PCR sensitivity.
6. Specificity test
The specificity test of real-time fluorescence PCR detection of Xinjiang isolate of apricot chlorotic leaf roll phytoplasma is carried out by taking total DNA of plant strains with apricot chlorotic leaf roll disease, jujube witches broom disease, grape golden yellowing, alfalfa witches broom disease, paulownia witches broom disease and willow witches broom disease stored in a laboratory and healthy apricot genome DNA collected in the field as templates.
7. Repeatability test
For 3 standard plasmids (containing 2.8X 10 in the initial reaction system) with different concentrations4～2.8×106copy/mL) the detection was repeated 3 times using established real-time fluorescent PCR and the stability of the method was evaluated by calculating the mean, Standard Deviation (SD) and Coefficient of Variation (CV) of the cycle threshold (Ct). Coefficient of Variation (CV) = percentage of standard deviation of measurement result to its mean.
8. Results of the experiment
Plasmid diluted 10-fold in gradient was used as template, corresponding to a concentration of 2.8X 108About 2.8X 10 copies/mL, and the logarithm of the number of copies of the corresponding plasmid DNA is the X axis, with the Ct value obtained as the y axis, a standard curve was prepared (see FIG. 1). The amplification efficiency of the instrument automatically generated standard curve is 92.76%, the correlation coefficient is 0.995, and the standard curve equation is y = -3.476x + 40.882. As can be seen from the amplification curve, the lower limit of the detection sensitivity of the fluorescent PCR is 28 copies/mL, which is 100 times higher than that of the conventional PCR detection method (see FIG. 2 and FIG. 3). According to the lowest copy number detected, a judgment standard of a detection result is established: on the premise of no Ct value in negative, the Ct value of the sample to be detected is positive below 34, 34-40 is suspicious, the template amount needs to be increased for detection again, and the sample without the Ct value is negative.
The primer and the probe can specifically detect the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma, and the grape golden phytoplasma, the jujube witches disease phytoplasma and the alfalfa arbuscular phytoplasma of the elm etiolated group (16 Sr V) of the same genus phytoplasma, and the paulownia arbuscular phytoplasma, the willow arbuscular phytoplasma and the healthy apricot tree belonging to the phyroma emerald etiolated group (16 Sr I) are all negative (see figure 4). Therefore, the primer and the probe designed by the invention have stronger specificity to Xinjiang isolate of apricot chlorotic leafroll phytoplasma, can better distinguish the phytoplasma among different groups and in the groups, have better specificity, and after the specificity verification is passed, the primer and the probe can be applied to the qualitative detection of the Xinjiang isolate of apricot chlorotic leafroll phytoplasma.
The result of the repeatability test shows that (table 1), the error of the Ct value of each repeated test is less than 1 cycle, and the variation coefficient of the Ct value is less than 5%, which proves that the real-time fluorescence PCR system established by the research has good repeatability, thereby ensuring the reliability and stability of the detection result among different samples.
TABLE 1 repeatability tests for real-time fluorescent PCR
Example 3 application of Xinjiang isolate real-time fluorescent PCR detection method of apricot chlorotic leafroll phytoplasma
The collected suspected apricot chlorotic leaf roll disease samples showing different disease symptoms are detected by adopting the established real-time fluorescent quantitative PCR detection method for the Xinjiang isolate of the apricot chlorotic leaf roll phytoplasma. The specific implementation mode is as follows:
1. extraction of total DNA of suspected apricot chlorosis leaf curl disease plant
Extracting total DNA of apricot branch phloem by using Plant Genomic extraction Kit (Plant Genomic DNA Kit, TIANGEN), and storing in a refrigerator at-40 deg.C for use.
2. Real-time fluorescent quantitative PCR detection
The reaction system for real-time fluorescent quantitative PCR detection of Xinjiang isolate of apricot chlorotic leafroll phytoplasma is 25 muL: comprises 1.0 muL of DNA template, 2.5 muL of 10 XPCR Buffer and 25mm/uL MgCl23.2uL，2.5 mmol/L dNTPs 2.0 µL，2.5U/µL ExTaqUpstream primer ACLR-F10.6 muL of DNA polymerase 0.5 muL, 10 mumol/L, downstream primer ACLR-R10.6 muL of 10 mumol/L, TaqMan Probe ACLR-Probe 0.8 muL of 10 mumol/L, Bovine Serum Albumin (BSA) 5.0 muL of 1.0 mg/mL, complement sterilization ddH2O to 25 μ L. The fluorescent quantitative PCR reaction program is that the pre-denaturation is carried out for 5 min at the temperature of 95 ℃; denaturation at 95 deg.C for 15 s, annealing at 60 deg.C for 30 s, and extension at 72 deg.C for 20 sThe fluorescence was collected by setting FAM fluorescence channel at 72 ℃ for 40 cycles.
3. Determination of results
After the reaction is finished, the Ct value of each sample is automatically given by the instrument, the Ct value is recorded, and the detection result is analyzed according to the result judgment standard established in the example 2 and the fluorescence quantitative PCR standard curve equation of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma.
4. Results of sample testing
The disease symptoms were detected as half-leaf curl, whole leaf curl, and asymptomatic (grade 0) diseased plants, respectively, according to the above-described method. The results show that the real-time fluorescent quantitative PCR detection results of half-leaf curl, whole leaf curl and asymptomatic disease strains are positive reactions, and the Ct values are 26.16, 23.25 and 29.69 in sequence. Calculating the copy numbers of the phytoplasma 16S rDNA fragment in the branches and leaves of the apricot trees with half leaf curl and whole leaf curl to be 8.2 multiplied by 10 respectively according to the standard curve equation of the fluorescence quantitative PCR of the Xinjiang isolate of the apricot chlorotic leaf phytoplasma and the Xinjiang isolate established in the example 24Copy/g fresh weight, 7.4X 105Copy/g fresh weight, copy number of phytoplasma 16SrDNA fragment in asymptomatic branches and leaves reaches 1.6 multiplied by 103Copies/g fresh weight.
Sequence listing
<110> Sinkiang university of agriculture
<120> qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma
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<213>apricot chlorotic leaf roll phytoplasma
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gaaacgcagt gcagcttgca tgcctgcagg tcgacgattc atgcaagtcg aacggagacc 60
cctcaaaagg tcttagtggc gaacgggtga gtaacacgta agtaacctac ctttaagacg 120
aggataacaa tcggaaacgg ttgctaagac tggataggaa ataaaaaggc atctttttgt 180
ttttaaaaga ccttcttcgg agggtatgct taaagagggg ctcgcgccac attagttagt 240
tggtgaggta aaggcttacc aagattatga tgtgtagctg gactgagagg ttgaacagcc 300
acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aattttcggc 360
aatggaggaa actctgaccg agcgacgccg cgtgaacgat gaagtatttc ggtatgtaaa 420
gttcttttat tgaacaagaa aaaatagtgg aaaaactatc ttgacgttat tcaatgaata 480
agccccggct aactatgtgc cagcagccgc ggtaagacat agggggcgag cgttatccgg 540
aattattgag cgtaaagggt gcgtaggcgg ttagataagt ctataattta atttcagtgc 600
ttaacgctgt cttgttatag aaactgtctt gactagagtg agatagaggc aagcggaatt 660
ccatgtgtag cggtaaaatg tgtaaatata tggaggaaca ccagaagcgt aggcggcttg 720
ctgggtcttt actgacgctg aggcacgaaa gcgtggggag caaacaggat tagataccct 780
ggtagtccac gctgtagacg atgagtacta agtgtcgggg caactcggta ctgaagttaa 840
cacattaagt actccgcctg agtagtacgt acgcaagtat gaaacttaaa ggaattgacg 900
ggactccgca caagcggtgg atcatgttgt ttaattcgaa ggtacacgaa aaaccttacc 960
aggtcttgac atactctgcg aagctataga aatatagtgg aggttatcag ggatacaggt 1020
ggtgcatggt tgtcgtcagt tcgtgtcgtg agatgttagg ttaagtccta aaacgaacgc 1080
aacccctgtc gttagttacc agcacgtaat ggtggggact ttagcgagac tgccaattaa 1140
acattggagg aaggtgggga taacgtcaaa tcatcatgcc ccttatgatc tgggctacaa 1200
acgtgataca atggctgtta caaagagtag ctgaaacgcg agtttttagc caatctcaaa 1260
aagacagtct tagtccggat tgaagtctgc aactcgactt catgaagctg gaatcgctag 1320
taatcgcgaa tcagcatgtc gcggtgaata cgttctcggg gtttgtacac accgcccgtc 1380
aaaccacgaa agttagcaat acccgaaagc agtggcttaa cttcgcaaga agagggagct 1440
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Claims (6)
1. A qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll mycoplasma is characterized in that the oligonucleotide sequences of the pair of specific PCR primers and the fluorescence Taqman probe are an upstream primer ACLR-F1: 5'-CCCTGGTAGTCCACGCTGTAG-3'; a downstream primer ACLR-R1: 5'-AGACCTGGTAAGGTTTTTCGTGTAC-3'; 5'-TTAATGTGTTAACTTCAGTACCG-3' for the Probe ACLR-Probe; wherein the size of the amplified fragment of the primer pair is 190 bp; the 5 'end of the probe is provided with a FAM marker of a reporter fluorescent dye, and the 3' end of the probe is provided with a TAMRA marker of a quenching fluorescent dye.
2. A qualitative and quantitative detection method for Xinjiang isolate of apricot chlorotic leafroll phytoplasma comprises the following steps:
(1) extracting the total DNA of the phloem or vein of the branches of the apricot trees by adopting a plant genome extraction kit, and storing the apricot trees in a refrigerator at the temperature of minus 40 ℃ for later use;
(2) taking the extracted total DNA of the plant as a template, respectively setting a positive control, a healthy plant total DNA control and a sterile double distilled water control, and carrying out real-time fluorescent quantitative PCR detection by adopting an ACLR-Probe Probe and an ACLR-F1/ACLR-1 primer pair;
(3) according to the reaction Ct value of each sample, judging whether the Xinjiang separator of the apricot chlorotic leafroll has the Xinjiang separator of the apricot chlorotic leafroll; and calculating the copy concentration of the Xinjiang isolate 16S rDNA gene fragment of the apricot chlorotic leafroll phytoplasma in the detected sample through a pre-established standard curve equation of fluorescent quantitative PCR reaction, thereby obtaining the content of the Xinjiang isolate of the apricot chlorotic leafroll phytoplasma in the sample and further realizing quantitative detection.
3. The qualitative and quantitative detection method for Xinjiang isolates of apricot chlorotic leafroll phytoplasma according to claim 2, wherein the reaction system for real-time fluorescent quantitative PCR detection is 25 μ L: comprises 1.0 muL of DNA template, 2.5 muL of 10 XPCRBuffer and 25 mm/muL MgCl23.2 µL，2.5 mmol/L dNTPs 2.0 µL，2.5 U/µL ExTaqUpstream primer ACLR-F10.6 muL of DNA polymerase 0.5 muL, 10 mumol/L, downstream primer ACLR-R10.6 muL of 10 mumol/L, TaqMan Probe ACLR-Probe 0.8 muL of 10 mumol/L, bovine serum albumin 5.0 muL of 1.0 mg/mL, complement sterilization ddH2O to 25 µL。
4. The method for the qualitative and quantitative detection of Xinjiang isolate of Phytoplasma pruriens as claimed in claim 2, wherein the real-time fluorescent quantitative PCR detection is performed by pre-denaturing at 95 ℃ for 5 min; denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 20 s for 40 cycles, and fluorescence collection at 72 ℃ by setting FAM fluorescence channel.
5. The method for the qualitative and quantitative detection of the Xinjiang isolate of apricot chlorotic leafroll mycoplasma according to claim 2, wherein the positive determination criteria of the real-time fluorescent quantitative PCR detection result of the Xinjiang isolate of apricot chlorotic leafroll mycoplasma are as follows: if the Ct value of the sample fluorescence PCR amplification curve is less than or equal to 34 and the negative control and the blank control have no amplification curve, the sample to be detected contains the Xinjiang separator of the apricot chlorotic leafroll phytoplasma, otherwise, the sample does not contain the Xinjiang separator of the apricot chlorotic leafroll phytoplasma.
6. The method for the qualitative and quantitative detection of Xinjiang isolate of Phytoplasma pruriens as claimed in claim 2, wherein the standard curve equation of the fluorescence quantitative PCR reaction is as follows: y = -3.476x +40.882, wherein x is the logarithm of the copy number of Xinjiang isolate 16S rDNA gene fragment of apricot chlorotic leafroll mycoplasma in the tested sample, and y is a Ct value.
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