CN117778632B - Fluorescent quantitative PCR (polymerase chain reaction) detection primer, kit and method for sugarcane strip spot virus - Google Patents
Fluorescent quantitative PCR (polymerase chain reaction) detection primer, kit and method for sugarcane strip spot virus Download PDFInfo
- Publication number
- CN117778632B CN117778632B CN202410205828.4A CN202410205828A CN117778632B CN 117778632 B CN117778632 B CN 117778632B CN 202410205828 A CN202410205828 A CN 202410205828A CN 117778632 B CN117778632 B CN 117778632B
- Authority
- CN
- China
- Prior art keywords
- sstrv
- sugarcane
- quantitative pcr
- primer
- fluorescent quantitative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000000111 Saccharum officinarum Species 0.000 title claims abstract description 58
- 235000007201 Saccharum officinarum Nutrition 0.000 title claims abstract description 58
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 43
- 241000700605 Viruses Species 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title abstract description 23
- 238000003752 polymerase chain reaction Methods 0.000 title description 18
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 9
- 239000013612 plasmid Substances 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 7
- 239000013642 negative control Substances 0.000 claims description 6
- 239000012154 double-distilled water Substances 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000013641 positive control Substances 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 108020004414 DNA Proteins 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 241000723806 Sugarcane mosaic virus Species 0.000 description 9
- 241000357694 Sugarcane yellow leaf virus Species 0.000 description 9
- 241000522618 Sugarcane streak mosaic virus Species 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 241001455595 Sorghum mosaic virus Species 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000450599 DNA viruses Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000702463 Geminiviridae Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 108091036078 conserved sequence Proteins 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000010206 sensitivity analysis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000776471 DPANN group Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010025421 Macule Diseases 0.000 description 1
- 241000702459 Mastrevirus Species 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 241000724803 Sugarcane bacilliform virus Species 0.000 description 1
- 241000702287 Sugarcane streak virus Species 0.000 description 1
- 241001479213 Sugarcane striate virus Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a fluorescent quantitative PCR detection primer, a kit and a method for sugarcane site virus, wherein a primer pair comprises an upstream primer SStrV-F1 and a downstream primer SStrV-R1, the sequences of the primer pair are shown as SEQ.ID.NO.1 and SEQ.ID.NO.2 in a sequence table, and a fluorescent quantitative PCR detection method for sugarcane site virus is established according to the primer pair, and the method has good specificity, sensitivity and repeatability and can accurately detect SStrV virus.
Description
Technical Field
The invention relates to the technical field of virus nucleic acid detection, in particular to a fluorescent quantitative PCR detection primer, a kit and a method for sugarcane strip spot virus.
Background
Sugarcane (Saccharum officinarum l.) is a perennial sugar-producing crop of the family poaceae, the world's main sugar crops, cash crops and important energy crops, and also is an important sugar crop and cash crop in our country. In 2022, the planting area of sugarcane in China is 1686.80 mu, the yield of the sugarcane is 897 ten thousand tons, and the yield of the sugarcane accounts for 87.96% of the total yield of the sugar in China. Sugarcane is a vegetative crop, which is infected by various viruses during growth. With the development of the sugarcane industry, the influence of virus diseases on sugarcane yield is increasingly serious. Currently, viruses that harm sugarcane can be classified into DNA viruses and RNA viruses according to genetic materials. RNA viruses are mainly sugarcane mosaic virus (Sugarcane mosaic virus, SCMV), sorghum mosaic virus (Sorghum mosaic virus, srMV), sugarcane streak mosaic virus (Sugarcane streak mosaic disease virus, SCSMV) and sugarcane yellow leaf virus (Sugarcane yellow leaf virus, SCYLV), and DNA viruses are mainly sugarcane baculovirus (Sugarcane bacilliform virus, SCBV) and sugarcane streak virus (Sugarcane striate virus, SStrV). Sugarcane strip spot virus is a new virus found in 2015, which was first found in sugarcane germplasm resources in florida, citruliprop, france and wankelian islands; it belongs to the genus Zea (Mastrevirus) of the Geminiviridae (GEMINIVIRIDAE) and has a circular single-stranded DNA monomer genome of about 2.7 and kb. SStrV after the sugarcane is infected, the sugarcane shows symptoms such as white spots, fading stripes or short sections and the like.
Yinfu Lin and the like are used for researching the occurrence, geographical distribution and genetic diversity of sugarcane stripe viruses in China, and the average incidence rate of SStrV in sugarcane areas in China is found to be 25.1%, and SStrV occur in Guangxi, yunnan, guangdong, hainan, fujian and other places, and the disease rate detected in the Guangdong sugarcane area SStrV is even as high as 90.3%. It can be seen that SStrV has been common in sugarcane areas in China. Currently, the host range of SStrV includes sugar cane, fruit cane and cane related germplasm resources (large stem wild species, dense cut hands, bamboo cane, macula and cane). Phylogenetic tree constructed from the full-length genome of SStrV isolates showed that SStrV isolates were divided into four different groups: a, B, C and D. Chinese isolates a and B consisted of, but did not include, groups C and D. Group SStrV A is the most prominent and most widely distributed strain (accounting for 98.7%) in China, and is divided into 8 subgroups, wherein the subgroup I has the most advantage and is most widely distributed in 8 subspecies.
At present, the research report of SStrV is few, only a few reports on SStrV occurrence, geographical distribution and genetic diversity are provided, and the origin of the virus population and the mechanism, molecular evolution mechanism, propagation mediator and the like of epidemic occurrence of the virus population are not clear. Meanwhile, the SStrV detection method is single, only one Polymerase Chain Reaction (PCR) is adopted, and the conventional PCR is simple to operate, but only can be qualitative and not quantitative, has low sensitivity, and has poor detection effect on samples with low virus content. More detection methods need to be developed to identify SStrV.
Disclosure of Invention
The whole genome sequence of the reported SStrV isolate is analyzed, a specific primer is designed according to a conserved sequence, and a fluorescence quantitative PCR (qPCR) detection method of SStrV is established. The method is applied to the research to identify SStrV loading amounts in tissues of different parts of sugarcane.
Specifically:
the fluorescent quantitative PCR detection primer for the sugarcane site virus comprises:
upstream primer SStrV-F1:5'-CTTAGGGGAACTCGTTCGGG-3';
downstream primer SStrV-R1:5'-CGATTCCACCCTTCCTCACC-3'.
The PCR detection primer is applied to sugarcane site virus detection.
Wherein, the reaction conditions of the amplification are as follows: pre-denaturing at 95 ℃ for 3min, and amplifying by 40 cycles, wherein each cycle of denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30s.
Wherein the amplification template is obtained from +4 leaves of sugarcane.
The fluorescent quantitative PCR detection kit for the sugarcane site virus comprises the following components:
upstream primer SStrV-F1:5'-CTTAGGGGAACTCGTTCGGG-3';
downstream primer SStrV-R1:5'-CGATTCCACCCTTCCTCACC-3'.
The kit comprises the following reagents: positive control standard, PCR reaction solution and negative control;
The positive control standard substance is a recombinant plasmid containing a sugarcane point virus gene sequence, the PCR reaction liquid comprises an upstream primer SStrV-F1, a downstream primer SStrV-R1 and 2X Taq plus Master Mix, and the negative control substance is double distilled water.
Wherein, the recombinant plasmid takes pMD19-T as a vector.
The beneficial effects of the invention are as follows:
The analysis of the whole genome sequence of the SStrV isolates is carried out, specific primers are designed according to the conserved sequence, a fluorescence quantitative PCR (qPCR) detection method of SStrV is established, the optimized reaction conditions are adopted, the logarithm of the Cq value and the copy number of the concentration of the standard substance in the drawn standard curve is in a good linear relation, the peak value of the melting curve is single, the qPCR detection method has good specificity, SStrV can be specifically detected, and SCBV, SCMV, SCSMV, srMV, SCYLV and other sugarcane viruses can not be detected. The sensitivity test showed that the sensitivity of the established SStrV fluorescent quantitative PCR method was 100 times that of the conventional PCR method. The repeated test results of the intra-group and inter-group show that the repeated variation coefficient of the intra-group and inter-group is less than 1%, which indicates that the established SStrV fluorescent quantitative PCR method has good repeatability. In conclusion, the SStrV fluorescent quantitative PCR method established by the research has the advantages of high specificity, high sensitivity, good repeatability and the like, and can be used for accurately detecting SStrV viruses.
The present study identified the loadings of the different tissue sites SStrV of 8 sugarcane varieties using the established SStrV fluorescent quantitative PCR assay, and found that the loading of SStrV was significantly different among the 8 sugarcane varieties, which may be related to the characteristics of the sugarcane varieties themselves. Of the 7 tissue sites (-1 leaf, +1 leaf, +2 leaf, +4 leaf, root, stem and bud) of the 8 sugarcane varieties, the SStrV load of +4 leaf was the highest, reaching significant differences from the other tissue sites. Second, the load of SStrV in +2 leaves is also higher.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 shows the result of SStrV fragment PCR amplification, wherein the lanes are M: markerDS2000,1-2 amplified SStrV mesh fragment, 3: a negative control;
FIG. 2 is a standard curve of the fluorescent quantitative PCR of the present invention constructed from plasmid DNA standards having copy numbers of 1.31X10 8 copies/. Mu. L SStrv, respectively, by 10-fold gradient dilution;
FIG. 3 is an amplification curve of the fluorescent quantitative PCR of the present invention, constructed from left to right by 10-fold gradient dilution of plasmid DNA standards having copy numbers of 1.31X10 8 copies/. Mu. L SStrv, respectively;
FIG. 4 is a dissolution curve of the fluorescent quantitative PCR of the present invention, with a peak corresponding temperature greater than 80 ℃; FIG. 5 is a fluorescent quantitative PCR-specific experimental amplification curve of the present invention, 1: SStrV,2 to 6: SCBV, SCMV, SCSMV, srMV, SCYLV,3: blank control;
FIG. 6 is a plot of amplification for fluorescent quantitative PCR sensitivity experiments of the present invention, constructed from 10-fold gradient dilutions of plasmid DNA standard with copy number 1.31X10 8 copies/. Mu. L SStrv, respectively, from left to right;
FIG. 7 is a sensitivity analysis of a conventional PCR detection method of the present invention, wherein M: markerDS2000, 1-8: 1.31X10 8 copies/. Mu.L-1.31X101 copies/. Mu.L, 9: blank control;
fig. 8 and 9 are results of detection of eight different tissue sites SStrV of the strain, where the error bars represent standard deviations and the lower case letters indicate that the viral SStrV loading differences in the different tissue sites reached significant levels (P < 0.05), calculated using the Duncan method.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
1. Materials and methods
1.1 Experimental materials
The 8 infected SStrV sugarcane germplasm used in the test is identified by the laboratory and stored in a sugarcane experimental base of Wenchang City, hainan province, and 5 sugarcane materials which are singly infected by sugarcane baculovirus (SCBV), sugarcane mosaic virus (SCMV), sugarcane streak mosaic virus (SCSMV), sorghum mosaic virus (SrMV) and sugarcane yellow leaf virus (SCYLV) are identified and stored by the laboratory.
1.2 Main reagent
DNA extraction reagents (CTAB, isopropanol, chloroform, ethanol, etc.), DH 5. Alpha. Competent cells, and 2X Taq plus Master Mix were all purchased from Beijing full-gold cloning Biotechnology Co., ltd., agarose gel DNA recovery kit (Hipure Gel Pure DNA MINI KIT) and 2X Q3 SYBR QPCR MQSTER Mix were all purchased from TOLOBIO, E.Z.N.ATM PLANT RNA KIT (RNA extraction) was purchased from Omoga, pMD19-T vector kit was purchased from Takara doctor Material technology (Beijing), and DNA MARKER was purchased from Guangdong biosciences, inc.
1.3 Primer design and Synthesis
According to the reported SStrV whole genome sequence, carrying out sequence alignment by DNAMAN 8 software, and analyzing the sequence of a conserved region; specific primers were designed in conserved regions using PRIMER PREMIER 5.0.0 software. qPCR specific primer sequence: SStrV-F1:5'-CTTAGGGGAACTCGTTCGGG-3', SStrV-R1:5'-CGATTCCACCCTTCCTCACC-3' the desired fragment size was expected to be 106bp. Conventional PCR specific primer sequences: SStrV-F2:5'-GCGAACCACGGAGGAGATAG-3', SStrV-R2:5'-CTCGCACGTATGCCTCTGAT-3' the desired fragment size was expected to be 410bp. Primers were synthesized by the division of biological engineering (Shanghai);
wherein the SStrV-F1 primer sequence is shown in SEQ.ID.NO. 1;
SStrV-R1 primer sequence is shown in SEQ ID.NO. 2;
SStrV-F2 primer sequence is shown in SEQ ID No. 3;
The SStrV-R2 primer sequence is shown as SEQ ID No. 4.
1.4 Extraction of viral nucleic acids
Taking 0.1g of sugarcane tissue sample, rapidly and fully grinding the sugarcane tissue sample in liquid nitrogen, and extracting total DNA of sugarcane by using a CTAB method; extracting total RNA of sugarcane by TRIzol method. The total RNA extracted was subjected to reverse transcription with REVERT AID FIRST-STRAND CDNA SYNTHESIS KIT of Thermo Scientific to obtain cDNA.
1.5 Preparation of recombinant plasmid Standard
Using SStrV positive sugarcane sample total DNA as a template, and using a primer pair SStrV-F3:5'-GAKGRCWTACTCACAGTCGAG-3', SStrV-R3:5'-GGMAASAATTGGTCGGTTGTC-3' is used as a specific primer for PCR amplification. The reaction was 25mL, 2X Taq plus Master Mix was 12.5mL, 1mL each of the upstream and downstream primers, 1mL of DNA template, and the total volume was made up to 25mL with ddH 2O. The reaction procedure is: 94 ℃ pre-denaturation for 5min,94 ℃ denaturation for 1min,52 ℃ annealing for 30s,72 ℃ extension for 1min,35 cycles, 72 ℃ extension for 10min, and the PCR amplification product is verified by agarose gel electrophoresis with the concentration of 1%. The fragment size of the PCR product is 1928bp, the target band is recovered by an agarose gel DNA recovery kit and then connected to a pMD19-T vector, and transformed to competent cells DH5 alpha, positive recombinant clones are screened out and sent to a biological engineering (Shanghai) stock company for sequencing, the sequencing correct recombinant plasmid pMD19-T-SStrV-qN is used as a positive plasmid standard substance, and the concentration of the positive plasmid standard substance is determined according to the formula: copy number= (concentration ng/. Mu.L. Times.6.02X10 23 copies/mol)/(recombinant plasmid length bp. Times.660 g/mol. Times.10 9), the copy number of the recombinant plasmid was calculated;
wherein the SStrV-F3 primer sequence is shown in SEQ.ID.NO. 5;
The SStrV-R3 primer sequence is shown as SEQ ID No. 6.
1.6 Establishment of fluorescent quantitative PCR method
1.6.1 Fluorescent quantitative PCR amplification system
The total volume of the PCR reaction was finally determined to be 20mL by optimizing the reaction system, wherein 2 XQ 3 SYBR QPCR MASTER Mix was 10mL, the upstream and downstream primers (concentration: 10 mmol/L) were each 0.4mL, the template was 2mL, and ddH2O was added to 20mL. Setting a temperature gradient to determine the optimal annealing temperature of the reaction, wherein the finally determined reaction conditions are as follows: pre-denaturing 95 ℃ to 3 min, and amplifying with 40 cycles, wherein each cycle denaturates 95 ℃ to 10 s, and anneals and extends 60 ℃ to 30 s.
1.6.2 Establishment of standard curve and sensitivity experiment
The recombinant plasmid is diluted 10 times by concentration gradient with sterilized ddH2O, 1.31X10 8 copies/. Mu.L to 1.31X10 2 copies/. Mu.L of plasmid with 6 gradients are selected as templates, the real-time fluorescence quantitative PCR amplification reaction is carried out by using the optimized system and the reaction conditions, the system automatically generates a standard curve after the reaction is finished, and the used primer pair is 1.3 part qPCR specific primer pair. Meanwhile, conventional PCR was performed using the same concentration of template, and sensitivity analysis was performed using a 1.3 part of conventional PCR-specific primer pair as the primer pair.
1.6.3 Specificity test
The specificity of the primers was verified by performing a real-time fluorescent quantitative PCR reaction using the DNA or cDNA of SStrV, SCBV, SCMV, SCSMV, srMV, SCYLV positive samples as templates, respectively, and simultaneously using sterilized ddH2O as a negative control for the test.
1.6.4 Repeatability test
And (3) performing repeated experiments in groups and repeated experiments among groups by using the established fluorescent quantitative PCR detection method and using recombinant plasmids with the same copy number as templates. The reproducibility within and between the fluorescent quantitative PCR groups was analyzed by calculating the Coefficient of Variation (CV) of the two groups of data.
1.7 Detection of different tissue parts SStrV of sugarcane
The amount of SStrV in the above tissue sites was determined by detection using the established fluorescent quantitative PCR method using 8 DNA of different tissue sites (-1 leaf, +1 leaf, +2 leaf, +4 leaf, root, stem and shoot) of the SStrV infected sugarcane germplasm as a template.
2. Results
2.1 Preparation of plasmid Standard
PCR amplification was performed using SStrV positive samples as templates and specific primer pairs SStrV-F3 and SStrV-R3 to obtain a target band of 1928bp in the expected size, and the results are shown in FIG. 1. The band was then recovered and purified, ligated with pMD19-T vector, transformed, single colonies were picked for sequencing, and plasmids were extracted from the single colonies with correct sequencing, designated pMD19-T-SStrV-qN. The concentration was 662.2 ng/mL, the ratio of OD260nm/OD280nm was 1.86, the purity was satisfactory (OD 260nm/OD280nm > 1.8), and the concentration was converted to a copy number of 1.31X 11 copies/. Mu.L.
2.2 Establishment of a Standard Curve
Diluting the recombinant plasmid according to a ratio of 10 times, selecting 6 gradient diluted recombinant plasmids as templates, and amplifying by using an optimized fluorescent quantitative PCR system and reaction conditions to obtain amplification curves corresponding to templates with different copy numbers (shown in figure 3); and drawing a standard curve by taking the logarithm of the template copy number as an abscissa (X axis) and the Cq value as an ordinate (Y axis). The results show that the method has good linear relationship between the Cq value and the logarithm of the copy number of the standard concentration at the template quantity of 1.31X10 3 to 1.31X10 8 copies/. Mu.L, wherein the correlation coefficient (r 2) is 0.999, the slope is-3.337, and the standard curve equation is: y= -3.337 x + 38.197 (as shown in fig. 2). The melting curve established in the experiment shows that all positive recombinant plasmids have unique specific peaks (shown in figure 4) corresponding to the melting curve, and the temperature corresponding to the peaks is 82.0 ℃, which indicates that primer dimers and non-specific amplified products are not generated in the experimental process.
2.3 Specificity test
The DNA or cDNA of SStrV, SCBV, SCMV, SCSMV, srMV, SCYLV positive samples is used as a template, the optimized real-time fluorescent quantitative PCR system is used as a reaction condition for amplification reaction, the result shows that the Cq values corresponding to other samples except SStrV positive samples when the other samples peak are all more than 35, and the system has a small amount of negligible reaction, and the result is shown in figure 5.
2.4 Sensitivity test
Carrying out 10-time gradient dilution on SStrV recombinant plasmids, selecting 8 gradients from 1.31X10 8 copies/. Mu.L to 1.31X10 1 copies/. Mu.L, and respectively carrying out real-time fluorescence quantitative PCR and conventional PCR under optimized reaction conditions by taking the diluted plasmids as templates. The results show that real-time fluorescence quantitative PCR can detect a minimum of 1.31X10. 10 1 copies/. Mu.L of standard, and the results are shown in FIG. 6;
Whereas the standard at a minimum of 1.31X10 3 copies/. Mu.L can be detected by conventional PCR, the results are shown in FIG. 7, and thus the sensitivity of the established fluorescent quantitative PCR method is 100 times that of the conventional PCR method.
2.5 Repeatability test
The established SStrV fluorescent quantitative PCR method was used to perform intra-group repeat test and inter-group repeat test, respectively, and the coefficient of variation was calculated, and the results are shown in Table 1. The result shows that the repeated variation coefficient in the group is 0.13-0.81%, the repeated variation coefficient between groups is 0.45-0.94%, and the variation coefficient of the two is less than 1%, which indicates that the established fluorescent quantitative PCR system has good repeatability in both single experiment and multiple experiments.
TABLE 1 fluorescent quantitative PCR repeatability test results
2.6 Detection of different tissue parts SStrV of sugarcane
The capacity of SStrV in the different tissue sites (-1 leaf, +1 leaf, +2 leaf, +4 leaf, root, stem and shoot) of 8 infected SStrV sugarcane germplasm was measured using established fluorescent quantitative PCR methods. The results show that: SStrV can be detected in the tissue sites of-1 leaf, +1 leaf, +2 leaf, +4 leaf, root, stem and bud of sugarcane, the load of SStrV is greatly different in different tissue sites of sugarcane, the load of SStrV in +4 leaf is highest, and significant difference from other tissue sites is achieved, and the results are shown in fig. 8 and 9. Thus, in the test SStrV, the best sample collection site is the +4 leaf of sugarcane.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (6)
1. The application of the fluorescent quantitative PCR detection primer for the sugarcane point virus in the fluorescent quantitative PCR detection of the sugarcane point virus is characterized in that the fluorescent quantitative PCR detection primer for the sugarcane point virus comprises the following components:
upstream primer SStrV-F1:5'-CTTAGGGGAACTCGTTCGGG-3';
downstream primer SStrV-R1:5'-CGATTCCACCCTTCCTCACC-3'.
2. The use according to claim 1, wherein the reaction conditions for the amplification are: pre-denaturing at 95 ℃ for 3min, and amplifying by 40 cycles, wherein each cycle of denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30s.
3. The use according to claim 2, wherein the amplification template is taken from the leaf of sugarcane.
4. The application of the kit in fluorescence quantitative PCR detection of sugarcane site virus is characterized in that the kit comprises:
upstream primer SStrV-F1:5'-CTTAGGGGAACTCGTTCGGG-3';
downstream primer SStrV-R1:5'-CGATTCCACCCTTCCTCACC-3'.
5. The use according to claim 4, wherein the kit further comprises the following reagents: positive control standard, PCR reaction solution and negative control;
The positive control standard substance is a recombinant plasmid containing a sugarcane point virus gene sequence, the PCR reaction liquid comprises the upstream primer SStrV-F1, the downstream primer SStrV-R1 and 2 multiplied by Taq plus Master Mix, and the negative control substance is double distilled water.
6. The use according to claim 5, wherein the recombinant plasmid is a pMD19-T vector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410205828.4A CN117778632B (en) | 2024-02-26 | 2024-02-26 | Fluorescent quantitative PCR (polymerase chain reaction) detection primer, kit and method for sugarcane strip spot virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410205828.4A CN117778632B (en) | 2024-02-26 | 2024-02-26 | Fluorescent quantitative PCR (polymerase chain reaction) detection primer, kit and method for sugarcane strip spot virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117778632A CN117778632A (en) | 2024-03-29 |
| CN117778632B true CN117778632B (en) | 2024-04-26 |
Family
ID=90402054
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410205828.4A Active CN117778632B (en) | 2024-02-26 | 2024-02-26 | Fluorescent quantitative PCR (polymerase chain reaction) detection primer, kit and method for sugarcane strip spot virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117778632B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555859A (en) * | 2013-11-07 | 2014-02-05 | 福建农林大学 | Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting sugarcane streak mosaic virus |
| WO2014066481A1 (en) * | 2012-10-24 | 2014-05-01 | Syngenta Participations Ag | Methods and kits for detection of a pathogen in sugarcane |
| CN108728581A (en) * | 2018-06-26 | 2018-11-02 | 广西大学 | The multiplex RT-PCR method of 5 kinds of sugarcane diseases of detection and its primer and kit simultaneously |
-
2024
- 2024-02-26 CN CN202410205828.4A patent/CN117778632B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014066481A1 (en) * | 2012-10-24 | 2014-05-01 | Syngenta Participations Ag | Methods and kits for detection of a pathogen in sugarcane |
| CN103555859A (en) * | 2013-11-07 | 2014-02-05 | 福建农林大学 | Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting sugarcane streak mosaic virus |
| CN108728581A (en) * | 2018-06-26 | 2018-11-02 | 广西大学 | The multiplex RT-PCR method of 5 kinds of sugarcane diseases of detection and its primer and kit simultaneously |
Non-Patent Citations (3)
| Title |
|---|
| Incidence, Geographical Distribution, and Genetic Diversity of Sugarcane Striate Virus in Saccharum Species in China;Lin Yinfu 等;《Plant Disease》;20211118;第105卷(第11期);摘要，第3531-3537页，补充资料 * |
| Molecular detection of sugarcane striate virus and sugarcane white streak virus and their prevalence in the Miami World Collection of sugarcane and related grasses;Wardatou Boukari 等;《Plant Pathology》;20200418;第69卷(第6期);摘要，第1060-1069页 * |
| 甘蔗基因表达定量PCR分析中内参基因的选择;阙友雄 等;《热带作物学报》;20091231;第30卷(第3期);摘要，第274-278页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117778632A (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Rapid detection of tobacco mosaic virus using the reverse transcription loop-mediated isothermal amplification method | |
| CN113789413B (en) | Primer pair, probe, kit and method for simultaneously detecting five lily viruses | |
| CN110628945B (en) | Multiple PCR detection method and special kit for pepper yellow mottle virus and cucumber mosaic virus in pepper leaves | |
| CN117778632B (en) | Fluorescent quantitative PCR (polymerase chain reaction) detection primer, kit and method for sugarcane strip spot virus | |
| KR101459616B1 (en) | Primer set for detecting viruses in sweet potato and method for detecting simultaneously said viruses using the same | |
| CN112481417A (en) | Rapid detection method and kit for new coronavirus typing and mutation sites | |
| CN111378781A (en) | Molecular marker primer for quickly and efficiently identifying salt-tolerant gene SKC1 of rice and application | |
| CN116287475A (en) | Nucleic acid sequence combination, kit and application of brassica yellow virus RT-LAMP-CRISPR isothermal detection | |
| CN116479166A (en) | Internal reference gene for stable expression of verticillium dahliae, and primer and application thereof | |
| AU2021100028A4 (en) | One-step multiplex RT-PCR method for simultaneous detection of three pathogens of sugarcane mosaic disease | |
| CN111118221B (en) | RPA primer, probe and kit for detecting mosaic virus of cassava mosaic virus of Sri Lanka | |
| CN112662816B (en) | Primer-probe combination, kit and method for detection of Amapari virus, Copiceus virus and Epimeris virus | |
| CN113667776A (en) | Real-time fluorescent quantitative PCR (polymerase chain reaction) method for detecting plantain mosaic virus in lily | |
| EP0785281B1 (en) | Genetic variety identifying method in hop | |
| CN105112534B (en) | Primer pair and method for identifying copy numbers of internal and external genes of chrysanthemum through fluorescent quantitative PCR | |
| KR101994831B1 (en) | Multiplex primer sets for determining genotype of locus resistant to Citrus tristeza virus and uses thereof | |
| CN116676424A (en) | qRT-PCR kit for detecting sweet potato virus H | |
| CN114045361A (en) | Multiple RT-PCR method for simultaneously detecting 4 tobacco viruses | |
| CN116987820A (en) | qRT-PCR kit for detecting sweet potato virus E | |
| CN112662817B (en) | Primer probes, target combinations, kits and methods for detecting Latinovirus, Mobala virus and Mopeya virus | |
| CN116397040B (en) | Single copy papaya gene and method for detecting copy number of exogenous gene in transgenic papaya by using same | |
| KR102625086B1 (en) | Biomarkers for determining the maturation date of wheat | |
| JP6220332B2 (en) | Hop variety identification method | |
| CN110079626B (en) | SSR primers for curvularia lunata genetic diversity and genetic relationship analysis and application | |
| CN112980985B (en) | PCR primer group for identifying or screening cabbage hybrid lethal parent type 1 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |