CN110951924A - One-step multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease - Google Patents
One-step multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease Download PDFInfo
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Abstract
A one-step method multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease establishes a multiple RT-PCR detection method which can simultaneously complete reverse transcription and PCR amplification of 3 pathogens in the same reaction tube under the same reaction condition by optimizing specific primers of sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus, concentration, enzyme dosage, annealing temperature and the like through one RT-PCR reaction, has simple operation steps, and avoids repeated detection of conventional RT-PCR or two-step method multiple RT-PCR reaction steps, more complex procedures, long time consumption and cross contamination. The method has the characteristics of simplicity, convenience, high efficiency, strong specificity, high sensitivity and the like, can be used for production and application of virus-free healthy sugarcane seedlings, germplasm exchange and introduction quarantine, and has important significance for preventing and controlling diffusion and spread of sugarcane mosaic disease.
Description
Technical Field
The invention belongs to the technical field of plant virus detection, and particularly relates to a one-step multiple RT-PCR detection method for simultaneously detecting sugarcane mosaic virus, sugarcane mosaic virus and 3 pathogens of sugarcane mosaic virus.
Background
The sugarcane is the most important sugar crop in China, the sugar yield of the sugarcane accounts for more than 85% of the total sugar yield of the whole country, and the sugarcane plays an important role in agricultural economic income. Sugarcane mosaic disease is worldwide sugarcane cancer caused by virus, which causes the quality of infected plants to be deteriorated, the germination rate of seedlings to be reduced, tillering to be reduced, the crystallization rate of cane sugar to be reduced, the yield loss is 30-50% when the infection rate reaches more than 30%, the cane sugar content is reduced by 6-14%, the sugarcane mosaic disease becomes one of the diseases which are the most widely distributed and seriously damaged in sugarcane areas in China, hundreds of millions of economic losses are caused every year, and the continuous and stable development of the sucrose industry in China is severely restricted. At present, no effective medicament for preventing and controlling the sugarcane mosaic disease exists internationally, so that isolation quarantine, disease-resistant variety screening, non-toxic seedling cultivation and the like are important measures for preventing and controlling the sugarcane mosaic disease in a targeted manner, and rapid and accurate virus detection on seedlings and plants is particularly important.
Sugarcane mosaic disease etiology is complex and can be caused by a variety of viruses. The established Sugarcane mosaic disease pathogens in Sugarcane areas in China mainly include 3 Sugarcane streak mosaic virus (SCSMV), Sorghum mosaic virus (SrMV) and Sugarcane mosaic virus (SCMV). The symptoms caused by the 3 pathogenic viruses are very similar in expression, complex infection or cross infection phenomena often exist, the three pathogens are difficult to accurately distinguish only through the symptom expression, and the accurate distinction of the three pathogens is helpful for pertinently performing disease-resistant breeding and breeding resistant varieties of different virus pathogens; meanwhile, the damage of different virus pathogens in various regions can be accurately monitored, the reasonable distribution of varieties is realized, and a basis is provided for formulating scientific and effective sugarcane mosaic disease prevention and control measures; the virus-free healthy seedlings of the sugarcane and the virus-carrying conditions of imported and exported germplasms can be monitored in time, and the safety production of the sugarcane industry is guaranteed, so that accurate diagnosis must be carried out by a gene detection method.
The gene detection is to detect the virus from the nucleic acid level, the detection sensitivity and specificity are higher than those of serological detection, and the gene detection is particularly suitable for sample detection with high accuracy requirement. At present, the gene detection methods of sugarcane viruses mainly comprise PCR, RT-PCR, loop-mediated isothermal amplification technology and the like, wherein the conventional RT-PCR is the most widely applied RNA virus detection method at present, but the conventional RT-PCR can only detect a single virus at one time. For sugarcane mosaic disease which often occurs by complex infection of two or more pathogens in the field, the conventional RT-PCR is used for pathogen detection, 2 or 3 times of RT-PCR is needed, the operation steps are complicated, the used reagents are various, the cost is high, the result can be obtained only after at least 8 hours for detecting one sample, the time consumption is long, the detection efficiency is low, and the requirement of modern production cannot be met. The existing multiplex RT-PCR detection mostly adopts a two-step method, pathogen detection needs to be completed in two reaction systems, the reaction steps and the procedures are relatively complex, the time consumption is long, and cross contamination is easy to cause.
With the application of the sugarcane virus-free healthy seedlings in sugarcane production, a rapid, accurate and high-flux detection method for sugarcane mosaic disease pathogens is urgently needed, and no related report of one-step multiple RT-PCR detection for simultaneously detecting 3 sugarcane mosaic disease pathogens is found at present.
Disclosure of Invention
The invention aims to provide a one-step method multiple RT-PCR detection method for simultaneously detecting 3 sugarcane mosaic disease pathogens quickly, accurately and specifically, so as to simplify operation, reduce workload and improve efficiency.
The invention provides a one-step multiplex RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease, wherein the 3 pathogens of sugarcane mosaic disease are sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus, a sugarcane streak mosaic virus primer is adopted in the one-step multiplex RT-PCR detection method for detecting the sugarcane streak mosaic virus, the sugarcane streak mosaic virus primer consists of an SCSMV upstream primer and an SCSMV downstream primer, the length of a target fragment is 800bp, and the nucleotide sequence of the SCSMV upstream primer is as shown in SEQ ID NO: 1, the nucleotide sequence of the SCSMV downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the sorghum mosaic virus primer is used for detecting sorghum mosaic virus, the sorghum mosaic virus primer is composed of a SrMV upstream primer and a SrMV downstream primer, the length of a target fragment is 450bp, and the nucleotide sequence of the SrMV upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SrMV downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the sugarcane mosaic virus primer is used for detecting sugarcane mosaic virus, the sugarcane mosaic virus primer is composed of an SCMV upstream primer and an SCMV downstream primer, the length of a target fragment is 200bp, and the nucleotide sequence of the SCMV upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SCMV downstream primer is shown as SEQ ID NO: 6 is shown in the specification; and judging the detection result as follows:
when the detection sample is amplified to a single band with the size of 800bp, the sample is positive to the sugarcane streak mosaic virus; if no 800bp band exists, the sugarcane streak mosaic virus is negative;
when the detection sample is amplified to a single band with the size of 450bp, the sample is positive to sorghum mosaic virus; if the DNA fragment has no 450bp band, the DNA fragment is negative to sorghum mosaic virus;
when the detection sample is amplified to a single band with the size of 200bp, the sample is positive to the sugarcane mosaic virus; if no 200bp strip exists, the sugarcane mosaic virus is negative;
when the detection sample is amplified to the three bands simultaneously or any two bands simultaneously, the sample is infected with the corresponding virus.
The one-step method multiple RT-PCR detection method is characterized in that sterilization ddH is contained in a 20 mu L one-step method multiple RT-PCR reaction system2O8.4. mu.L, 2X 1Step Buffer 8.0. mu.L, 5U/. mu.L PrimeScript 1Step Enzyme Mix 0.8. mu.L, 20. mu.mol/L SCSMV upstream and downstream primers0.25 mu L of SrMV upstream primer and SrMV downstream primer of 20 mu mol/L, 0.4 mu L of SrMV upstream primer and LSCMV downstream primer of 20 mu mol/L and RNA template of 1.0 mu L; the one-step method multiple RT-PCR reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃, and pre-denaturation is carried out for 5min at 94 ℃; then denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; finally, extending for 10min at 72 ℃, and cooling to 12 ℃ to finish the reaction.
The invention provides a group of specific primer groups for simultaneously detecting 3 pathogens of sugarcane mosaic disease by adopting a one-step method multiple RT-PCR detection method, which consists of a sugarcane streak mosaic virus primer, a sorghum mosaic virus primer and a sugarcane mosaic virus primer, wherein the sugarcane streak mosaic virus primer is used for detecting the sugarcane streak mosaic virus, the sugarcane streak mosaic virus primer consists of an SCSMV upstream primer and an SCSMV downstream primer, the length of a target fragment is 800bp, and the nucleotide sequence of the SCSMV upstream primer is as shown in SEQ ID NO: 1, the nucleotide sequence of the SCSMV downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the sorghum mosaic virus primer is used for detecting sorghum mosaic virus, the sorghum mosaic virus primer consists of an SrMV upstream primer and an SrMV downstream primer, the length of a target fragment is 450bp, and the nucleotide sequence of the SrMV upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SrMV downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the sugarcane mosaic virus primer is used for detecting sugarcane mosaic virus, the sugarcane mosaic virus primer consists of an SCMV upstream primer and an SCMV downstream primer, the length of a target fragment is 200bp, and the nucleotide sequence of the SCMV upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SCMV downstream primer is shown as SEQ ID NO: and 6.
The one-step multiplex RT-PCR detection kit for simultaneously detecting 3 pathogens of sugarcane mosaic disease provided by the invention comprises a specific primer group for simultaneously detecting 3 pathogens of sugarcane mosaic disease by adopting the one-step multiplex RT-PCR detection method, wherein the 3 pathogens of sugarcane mosaic disease are sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus.
Further, the kit also comprises a 20 mu L one-step method multiplex RT-PCR reaction system: sterilization ddH2O8.4. mu.L, 2X 1Step Buffer 8.0. mu.L, 5U/. mu.L PrimeScript 1Step Enzyme Mix 0.8. mu.L, 20. mu. mol/LSCSMV upstream and downstream introduction0.25. mu.L of each substance, 0.4. mu.L of each of 20. mu. mol/L SrMV upstream and downstream primers, 0.25. mu.L of each of 20. mu. mol/L SCMV upstream and downstream primers, and 1.0. mu.L of RNA template.
The invention also provides application of the specific primer group or the kit in simultaneous detection of 3 sugarcane mosaic diseases by using a one-step multiplex RT-PCR method, wherein the 3 pathogens are sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention can complete reverse transcription and PCR amplification by one RT-PCR reaction, can simultaneously amplify specific bands of 3 pathogens causing sugarcane mosaic disease, and has strong specificity, rapidness, accuracy, simple operation steps and high sensitivity.
The invention respectively designs and synthesizes three pairs of corresponding specific primers aiming at coat protein gene nucleotide sequences of 3 pathogens of sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus, and can complete reverse transcription and PCR amplification by taking total RNA of a sample as a template in one RT-PCR reaction system and the same reaction tube under the same reaction condition through optimizing the three pairs of specific primers, the concentration of the specific primers, the enzyme dosage, the annealing temperature and the like in one step, thereby overcoming the defects that 2 or 3 times of RT-PCR is needed for pathogen detection of conventional RT-PCR, the operation steps are complicated, the types of reagents are multiple, the cost is high, the two-step method multiple RT-PCR reaction steps are high, the program is complex, the time consumption is long, and the cross contamination is caused.
The invention can make the SCSMV, SrMV and SCMV three pathogenic viruses still amplify corresponding specific bands when the concentration is 0.1 ng/mu L, namely, the invention has high detection sensitivity.
The invention can obtain the detection result only in 3 hours, greatly saves the detection time and improves the detection efficiency.
All reagents are added into the reaction tube before reaction, so that the pollution caused by adding reaction liquid after the amplification is avoided, and the occurrence of false positive is greatly reduced.
The method of the invention does not need a large amount of reagents and consumables in the whole detection process, thereby reducing the detection cost.
2. The method can quickly and accurately detect and distinguish three pathogens causing the sugarcane mosaic disease, can be used for pertinently carrying out disease-resistant breeding on the sugarcane mosaic disease, accurately monitoring the occurrence and damage of different virus pathogens in various regions, realizing reasonable distribution of varieties, formulating scientific and effective sugarcane mosaic disease prevention and control measures, timely monitoring healthy sugarcane virus-free seedlings and the virus-carrying conditions of imported and exported germplasms, and has important significance for safe production, germplasm exchange, introduction and quarantine of the sugarcane industry and prevention and control of diffusion and spread of the sugarcane mosaic disease.
SEQ ID NO: 1 shows the nucleotide sequence of the SCSMV upstream primer.
SEQ ID NO: 2 shows the nucleotide sequence of the SCSMV downstream primer.
SEQ ID NO: 3 shows the nucleotide sequence of the SrMV upstream primer.
SEQ ID NO: 4 shows the nucleotide sequence of the SrMV downstream primer.
SEQ ID NO: shown in 5 is the nucleotide sequence of the SCMV forward primer.
SEQ ID NO: shown in figure 6 is the nucleotide sequence of the SCMV downstream primer.
Drawings
FIG. 1 is a specific electrophoresis diagram of 3 kinds of pathogenic primers RT-PCR detection, M is DNA Marker E; 1. 3, 5 are SCSMV, SrMV, SCMV in turn; 2. 4 and 6 are corresponding negative controls respectively.
FIG. 2 is an electrophoretogram optimized for the concentration of 3 kinds of pathogenic primers, M is DNA Marker E; 1-4 are sequentially a first group, a second group, a third group and a fourth group of primer combinations.
FIG. 3 is a DNA polymerase dosage optimization electropherogram, M is DNA Marker E; 1-4 are 2.5U, 4U, 5U and 6U in sequence.
FIG. 4 is an annealing temperature-optimized electropherogram, M being DNA Marker E; 1-8 are 50 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C, 62 deg.C, 64 deg.C in sequence.
FIG. 5 is a one-step multiplex RT-PCR specific and stability electrophoretogram, M is DNA Marker E; 1 is SCSMV + SrMV + SCMV; 2 is SCSMV + SrMV; 3 is SCSMV + SCMV; 4 is SrMV + SCMV; 5 is SCSMV; 6 is SrMV; 7 is SCMV; 8 is a negative control; blank control 9.
FIG. 6 is a one-step multiplex RT-PCR sensitivity analysis electrophoretogram, M is DNA Marker E; the dilution times of 1-7SCSMV, SrMV and SCMV 3 pathogenic virus composite RNA templates are 10 in sequence0、101、102、103、104、105、106。
Detailed Description
The present invention is further illustrated by the following specific examples, which are not specifically intended to be limiting of the conventional methods.
1. Primer design
According to the nucleotide sequences of coat protein genes of 3 pathogens of sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus, BLAST online primer design software is applied to design three pairs of specific primers, namely the sugarcane streak mosaic virus primer, the sorghum mosaic virus primer and the sugarcane mosaic virus primer, and homology and complementarity between the primers are analyzed and optimized to ensure that the three pathogens can amplify specific fragments with obvious differences, and finally, the primers are synthesized by Shanghai biological engineering Co., Ltd. The sugarcane streak mosaic virus primer consists of an SCSMV upstream primer and an SCSMV downstream primer, the length of a target fragment is 800bp, and the nucleotide sequence of the SCSMV upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SCSMV downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the sorghum mosaic virus primer is used for detecting sorghum mosaic virus, the sorghum mosaic virus primer is composed of a SrMV upstream primer and a SrMV downstream primer, the length of a target fragment is 450bp, and the nucleotide sequence of the SrMV upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SrMV downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the sugarcane mosaic virus primer is used for detecting sugarcane mosaic virus, the sugarcane mosaic virus primer is composed of an SCMV upstream primer and an SCMV downstream primer, the length of a target fragment is 200bp, and the nucleotide sequence of the SCMV upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SCMV downstream primer is shown as SEQ ID NO: and 6.
2. Total RNA extraction
Sugarcane leaves infected with sugarcane streak mosaic virus (SCSMV), sorghum mosaic virus (SrMV) and sugarcane mosaic virus (SCMV) individually are detected by the method of "Wang X Y, Li W F, Huang Y K, et al, molecular detection and viral assessment analysis of viral using biological systems in new sugarcane and biological in China, European Journal of Plant Pathology,2017,148(4):931 940", 0.2g of sugarcane leaves infected with SCSMV, SrMV, SCMV individually are collected, total RNA of leaves is extracted using a Plant genomic RNA extraction kit (exemplified by TransZol Plant RNA extraction kit from Beijing all-purpose gold Biotech), and the specific procedures are performed according to the description.
3. Single plex RT-PCR detection
3.1 one-step RT-PCR amplification
Sterilizing ddH in 20 mu L one-step RT-PCR reaction system27.6. mu.L of O, 8.0. mu.L of 2X 1Step Buffer, 1.0. mu.L of PrimeScript 1Step Enzyme Mix, 0.4. mu.L of 20. mu. mol/L of each of the upstream and downstream primers of SCSMV, 0.4. mu.L of each of the upstream and downstream primers of 20. mu. mol/L of LSrMV, 0.4. mu.L of each of the upstream and downstream primers of 20. mu. mol/L of SCMV, and 1.0. mu.L of RNA template. The one-step RT-PCR reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃, and pre-denaturation is carried out for 5min at 94 ℃; then denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; finally, extending for 10min at 72 ℃, and cooling to 12 ℃ to finish the reaction.
And (3) performing single RT-PCR detection by adopting the one-step RT-PCR reaction system and the one-step RT-PCR reaction condition and respectively taking the total RNAs respectively infected with the 3 viruses extracted in the step (2) as templates.
3.2 electrophoretic detection of PCR products
6. mu.L of the PCR amplification product was subjected to 1.5% agarose gel electrophoresis. The amplified bands of SCSMV, SrMV and SCMV were obtained in the order of 800bp, 450bp and 200bp, and the sizes thereof were consistent with theoretical values, and the results are shown in FIG. 1.
4. One-step method multiple RT-PCR optimization
4.1 optimization of primer concentration ratio
4 primer concentration combinations were set:
a first group: the final concentrations of 20. mu. mol/L of the SCSMV forward primer, 20. mu. mol/L of the SCSMV reverse primer, 20. mu. mol/L of the SrMV forward primer, 20. mu. mol/L of the SrMV reverse primer, 20. mu. mol/L of the SCMV forward primer and 20. mu. mol/L of the SCMV reverse primer are all 0.25. mu. mol/L.
Second group: the final concentrations of the 20. mu. mol/L SCSMV upstream primer and the 20. mu. mol/L SCSMV downstream primer are both 0.2. mu. mol/L, the final concentrations of the 20. mu. mol/L SrMV upstream primer and the 20. mu. mol/L SrMV downstream primer are both 0.25. mu. mol/L, and the final concentrations of the 20. mu. mol/L SCMV upstream primer and the 20. mu. mol/L SCMV downstream primer are both 0.4. mu. mol/L.
Third group: the final concentrations of the 20. mu. mol/L SCSMV upstream primer and the 20. mu. mol/L SCSMV downstream primer are both 0.2. mu. mol/L, the final concentrations of the 20. mu. mol/L SrMV upstream primer and the 20. mu. mol/L SrMV downstream primer are both 0.25. mu. mol/L, and the final concentrations of the 20. mu. mol/L SCMV upstream primer and the 20. mu. mol/L SCMV downstream primer are both 0.25. mu. mol/L.
And a fourth group: the final concentrations of the 20. mu. mol/L SCSMV upstream primer and the 20. mu. mol/L SCSMV downstream primer are both 0.25. mu. mol/L, the final concentrations of the 20. mu. mol/L SrMV upstream primer and the 20. mu. mol/L SrMV downstream primer are both 0.4. mu. mol/L, and the final concentrations of the 20. mu. mol/L SCMV upstream primer and the 20. mu. mol/L SCMV downstream primer are both 0.25. mu. mol/L.
According to the 3. single RT-PCR detection method, the multi-RT-PCR reaction is carried out by taking 3 kinds of pathogenic virus composite RNA as templates, and the electrophoresis result is shown in figure 2. Through screening, the optimal final concentrations of the 20 mu mol/L SCSMV upstream primer and the 20 mu mol/L SrMV upstream primer and the 20 mu mol/L SCMV downstream primer are respectively 0.25, 0.4 and 0.25 mu mol/L, namely 0.25 mu L of the 20 mu mol/L SCSMV upstream primer and the 20 mu mol/L SCSMV downstream primer in a 20 mu L one-step multiplex RT-PCR system, 0.4 mu L of the 20 mu mol/L SrMV upstream primer and the 20 mu mol/L SrMV downstream primer respectively, and 0.25 mu L of the 20 mu mol/L SCMV upstream primer and the 20 mu mol/L SCMV downstream primer respectively.
4.2 optimization of the amount of DNA polymerase used
4 gradients were set for the amount of Taq DNA polymerase at a concentration of 5U/. mu.L: 2.5U, 4U, 5U and 6U, performing multiplex RT-PCR reaction by using 3 kinds of pathogenic virus composite RNA as templates according to the 3. single RT-PCR detection method, and obtaining an electrophoresis result shown in figure 3. The bands were clear when 4U and 6U of Taq DNA polymerase was used, but from the viewpoint of cost saving, 4U was selected, i.e., 0.8. mu.L of PrimerScript 1Step Enzyme Mix.
4.3 annealing temperature optimization
The annealing temperature was optimized with a gradient from 50 ℃ to 64 ℃ every 2 ℃, and multiple RT-PCR reactions were performed using 3 pathogenic virus composite RNAs as templates according to the 3-singleplex RT-PCR detection method described above, and the electrophoresis results are shown in fig. 4. As can be seen from FIG. 4, 3 amplified bands at 62 ℃ are clear and have no impurity band, so the annealing temperature of 62 ℃ is selected as the best.
4.4 one-step multiplex RT-PCR specificity and stability
According to the optimization result, the optimal reaction system of the one-step method multiple RT-PCR is as follows: sterilization ddH2O8.4. mu.L, 2X 1Step Buffer 8.0. mu.L, 5U/. mu.L PrimerScript 1Step Enzyme Mix 0.8. mu.L, 20. mu. mol/L SCSMV forward primer 0.25. mu.L, 20. mu. mol/L SCSMV reverse primer 0.25. mu.L, 20. mu. mol/L SrMV forward primer 0.4. mu.L, 20. mu. mol/L LSrMV reverse primer 0.4. mu.L, 20. mu. mol/L SCMV forward primer 0.25. mu.L, 20. mu. mol/L SCMV reverse primer 0.25. mu.L, RNA template 1.0. mu.L; the optimal reaction conditions of the one-step method multiple RT-PCR are as follows: reverse transcription is carried out for 30min at 50 ℃, and pre-denaturation is carried out for 5min at 94 ℃; then denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; finally, extending for 10min at 72 ℃, and reducing the temperature to 12 ℃ to finish the reaction.
Specifically, multiple RNA samples which are independently infected with SCSMV, SrMV and SCMV are taken to prepare 7 different detection samples according to the modes of 3 pathogenic virus composite RNA templates (1 type), two pathogenic virus randomly combined composite RNA templates (3 types) and single pathogenic virus RNA template (3 types), the total RNA of the healthy sugarcane sample is used as negative control, and the 4.4 optimized conditions are adopted to detect and repeat experiments. The electrophoresis results are shown in FIG. 5. FIG. 5 shows that: 3 pairs of primers are mixed in the same reaction system, RNA of 3 viruses, 2 viruses and 1 virus is respectively used as templates, corresponding specific bands can be amplified simultaneously, no miscellaneous band appears, no amplification band appears in negative and blank control samples, the 3 pairs of primers are used for amplifying fragments of different viruses, the fragments are specific and specialized, and no cross and mutual interference phenomenon exists, and the established one-step method multiple RT-PCR can detect not only single pathogenic virus of the sugarcane mosaic disease, but also 3 pathogenic viruses of the sugarcane mosaic disease simultaneously.
5. Triple RT-PCR sensitivity assay
Taking SCSMV, SrMV and SCMV total RNA with the concentration of 100 ng/mu L respectively, mixing the three types of pathogenic virus total RNA with equal volume together to be used as a composite RNA template, and carrying out 10 steps on the composite RNA template0、101、102、103、104、105、106Multiple PCR amplifications were performed to detect sensitivity using 4.4 optimized conditions as described above with the results of electrophoresis shown in FIG. 6. As can be seen from FIG. 6, when the dilution ratio is 103When the RNA concentration of three pathogenic viruses, namely SCSMV, SrMV and SCMV, is 0.1 ng/mu L, the specific bands of the pathogenic viruses can still be amplified by the multiple RT-PCR, so that the invention has high sensitivity.
Sequence listing
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Claims (6)
1. A one-step multiplex RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease, wherein the 3 pathogens of sugarcane mosaic disease are sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus, and the method is characterized in that:
the one-step method multiple RT-PCR detection method adopts a sugarcane streak mosaic virus primer to detect the sugarcane streak mosaic virus, the sugarcane streak mosaic virus primer consists of an SCSMV upstream primer and an SCSMV downstream primer, the length of a target fragment is 800bp, and the nucleotide sequence of the SCSMV upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SCSMV downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the sorghum mosaic virus primer is used for detecting sorghum mosaic virus, the sorghum mosaic virus primer is composed of a SrMV upstream primer and a SrMV downstream primer, the length of a target fragment is 450bp, and the nucleotide sequence of the SrMV upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SrMV downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the sugarcane mosaic virus primer is used for detecting sugarcane mosaic virus, the sugarcane mosaic virus primer is composed of an SCMV upstream primer and an SCMV downstream primer, the length of a target fragment is 200bp, and the nucleotide sequence of the SCMV upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SCMV downstream primer is shown as SEQ ID NO: 6 is shown in the specification;
and judging the detection result as follows:
when the detection sample is amplified to a single band with the size of 800bp, the sample is positive to the sugarcane streak mosaic virus; if no 800bp band exists, the sugarcane streak mosaic virus is negative;
when the detection sample is amplified to a single band with the size of 450bp, the sample is positive to sorghum mosaic virus; if the DNA fragment has no 450bp band, the DNA fragment is negative to sorghum mosaic virus;
when the detection sample is amplified to a single band with the size of 200bp, the sample is positive to the sugarcane mosaic virus; if no 200bp strip exists, the sugarcane mosaic virus is negative;
when the detection sample is amplified to the three bands simultaneously or any two bands simultaneously, the sample is infected with the corresponding virus.
2. The one-step multiplex RT-PCR detection method according to claim 1, characterized in that: sterilization ddH is contained in a 20 mu L one-step method multiple RT-PCR reaction system2O8.4. mu.L, 2X 1Step Buffer 8.0. mu.L, 5U/. mu.L PrimeScript 1Step Enzyme Mix 0.8. mu.L, 20. mu. moL/L SCSMV upstream and downstream primers 0.25. mu.L, 20. mu. moL/L SrMV upstream and downstream primers 0.4. mu.L, 20. mu. moL/L SCMV upstream and downstream primers 0.25. mu.L, RNA template 1.0. mu.L; the one-step method multiple RT-PCR reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃, and pre-denaturation is carried out for 5min at 94 ℃; then denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; finally, extending for 10min at 72 ℃, and cooling to 12 ℃ to finish the reaction.
3. The specific primer group for simultaneously detecting 3 pathogens of sugarcane mosaic disease by adopting a one-step method multiple RT-PCR detection method is characterized in that: the kit comprises a sugarcane streak mosaic virus primer, a sorghum mosaic virus primer and a sugarcane mosaic virus primer, wherein the sugarcane streak mosaic virus primer is used for detecting the sugarcane streak mosaic virus, the sugarcane streak mosaic virus primer comprises an SCSMV upstream primer and an SCSMV downstream primer, the length of a target fragment is 800bp, and the nucleotide sequence of the SCSMV upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the SCSMV downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the sorghum mosaic virus primer is used for detecting sorghum mosaic virus, the sorghum mosaic virus primer consists of an SrMV upstream primer and an SrMV downstream primer, the length of a target fragment is 450bp, and the nucleotide sequence of the SrMV upstream primer is shown as SEQ ID NO: 3, the nucleotide sequence of the SrMV downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the sugarcane mosaic virus primer is used for detecting sugarcane mosaic virus, the sugarcane mosaic virus primer consists of an SCMV upstream primer and an SCMV downstream primer, the length of a target fragment is 200bp, and the nucleotide sequence of the SCMV upstream primer is shown as SEQ ID NO: 5, the nucleotide sequence of the SCMV downstream primer is shown as SEQ ID NO: and 6.
4. A one-step multiplex RT-PCR detection kit for simultaneously detecting 3 pathogens of sugarcane mosaic disease, which is characterized by comprising the specific primer group for simultaneously detecting 3 pathogens of sugarcane mosaic disease by the one-step multiplex RT-PCR detection method according to claim 3, wherein the 3 pathogens of sugarcane mosaic disease are sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus.
5. The kit of claim 4, further comprising a 20 μ L one-step multiplex RT-PCR reaction system: sterilization ddH2O8.4. mu.L, 2X 1Step Buffer 8.0. mu.L, 5U/. mu.L PrimeScript 1 StepEnzyme Mix 0.8. mu.L, 20. mu. mol/L of each of the upstream and downstream primers of SCSMV 0.25. mu.L, 20. mu. mol/L of each of the upstream and downstream primers of SrMV 0.4. mu.L, 20. mu. mol/L of each of the upstream and downstream primers of SCMV 0.25. mu.L, and 1.0. mu.L of RNA template.
6. Use of the specific primer set of claim 3 or the kit of any one of claims 4 to 5 for simultaneous detection of 3 pathogens of sugarcane mosaic disease, said 3 pathogens being sugarcane streak mosaic virus, sorghum mosaic virus and sugarcane mosaic virus.
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