CN113355436A - Primer and probe for detecting meloidogyne incognita by real-time fluorescence quantitative PCR (polymerase chain reaction) and application - Google Patents
Primer and probe for detecting meloidogyne incognita by real-time fluorescence quantitative PCR (polymerase chain reaction) and application Download PDFInfo
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- CN113355436A CN113355436A CN202110811251.8A CN202110811251A CN113355436A CN 113355436 A CN113355436 A CN 113355436A CN 202110811251 A CN202110811251 A CN 202110811251A CN 113355436 A CN113355436 A CN 113355436A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention belongs to the technical field of biology, and particularly relates to a primer and a probe for detecting meloidogyne incognita by real-time fluorescence quantitative PCR (polymerase chain reaction) and application of the primer and the probe. The invention relates to a primer and a probe for real-time fluorescent quantitative PCR detection of meloidogyne incognita, wherein a forward primer sequence Mi-F2:5'-CTACCCTTATCGGTGGATCACTA-3' and a reverse primer Mi-R2:5'-AATGACCCTGAACCAGACGTT-3' are adopted; the probe sequence Mi-P2: 5'-AGTTTGACCTCAATGCGGCCATTTGCGT-3'. Compared with the traditional morphological technology, the real-time fluorescence quantitative PCR method for detecting the meloidogyne incognita is convenient and efficient, and can realize high-specificity, high-sensitivity and high-flux rapid detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer and a probe for detecting meloidogyne incognita by real-time fluorescence quantitative PCR (polymerase chain reaction) and application of the primer and the probe.
Background
Root-knot nematodes (melodogyne spp.) are a class of plant root-system obligate endoparasitic nematodes that cause soil-borne diseases next to fungal diseases. The host range of the root-knot nematode is wide, more than 3000 plants can be infected, vegetables, fruits and grain crops are involved, the annual loss caused by the harm of the root-knot nematode in China exceeds 50 hundred million yuan, the economic loss is serious, and the healthy development of the agricultural industry in China is restricted. Therefore, the population density of the field nematodes is determined before the crops are sown or transplanted, and corresponding control strategies are formulated according to different population densities, so that the method has important significance for reducing damage loss.
In the prior art, a morphological identification method is adopted to identify the root-knot nematode, the method is used for inspecting by means of a microscope, the requirement on the professional knowledge background of a detector is high, and the detection speed is low.
Disclosure of Invention
The invention aims to provide a primer and probe composition for detecting meloidogyne incognita by real-time fluorescent quantitative PCR.
Still another object of the present invention is to provide the use of the primer probe composition.
It is a further object of the present invention to provide a kit comprising the above composition.
It is a further object of the present invention to provide a method for detecting meloidogyne incognita.
The primer and the probe for detecting the meloidogyne incognita by real-time fluorescence quantitative PCR according to the embodiment of the invention have the following nucleotide sequences:
the forward primer sequence Mi-F2:5'-CTACCCTTATCGGTGGATCACTA-3',
reverse primer sequence Mi-R2: 5'-AATGACCCTGAACCAGACGTT-3';
the probe sequence Mi-P2: 5'-AGTTTGACCTCAATGCGGCCATTTGCGT-3'.
According to the primer and the probe for real-time fluorescence quantitative PCR detection of Meloidogyne incognita according to the specific embodiment of the invention, the 5 'end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quenching group, wherein the fluorescent group can be selected from FAM, HEX, TET and the like, and the preferred scheme of the invention is FAM; the quenching group can be TAMRA, BHQ and the like, and the preferred embodiment of the invention is TAMRA.
The invention relates to application of a primer and a probe for detecting meloidogyne incognita by real-time fluorescence quantitative PCR, in particular to the aspect of detecting meloidogyne incognita.
According to the kit for detecting meloidogyne incognita, the kit comprises the primer and the probe, and the kit can be used for detecting meloidogyne incognita.
The method for detecting the meloidogyne incognita by real-time fluorescence quantitative PCR comprises the step of carrying out PCR amplification on DNA of the meloidogyne incognita by using the specific primer and the probe.
Wherein, the PCR reaction system comprises the following components: rTaq 0.5. mu. L, MgCl 23. mu.L, 10xPCR Buffer 2.5. mu. L, dNTPs 2. mu.L, probe 1. mu.L, primer 2. mu.L (1. mu.L each of Mi-F2 and Mi-R2), 0.1% BSA 5. mu. L, H2O4. mu.L, template 5. mu.L.
The PCR amplification procedure is as follows: 30s at 95 ℃; 5s at 94 ℃; 60 ℃ for 30s, 45 cycles.
The invention has the beneficial effects that:
the primer and the probe provided by the invention can specifically detect the meloidogyne incognita, and can not obtain positive results on other nematode DNA and soil-borne disease pathogen DNA serving as templates; the kit can realize the rapid quantitative detection of the meloidogyne incognita, has the detection sensitivity of 50 copies of the meloidogyne incognita DNA, and can detect the meloidogyne incognita DNA when the concentration of the meloidogyne incognita DNA is 1 multiplied by 101～1×108In the copy/mu L range, the linear relation is good, and the method can be used for accurate quantitative detection of qPCR.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the comparison result of the primers of the present invention and the existing primers for amplifying the genomic DNA of Meloidogyne incognita by qPCR (Taqman), wherein (1) the primers Mi-F2/R2 probe Mi-P2; (2) primer Mi-F3/R3; (3) the primer Mi-F4/R4 probes Mi-P2; (4) primer Mi-F5/R5; (5) primer RKNf/r;
FIG. 2 shows the results of experiments on the specificity of the primers and probes of the present invention;
FIG. 3 is a qPCR standard curve of Meloidogyne incognita.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1 design of primers
The invention designs the nucleotide sequence of the primer and the probe for detecting the meloidogyne incognita by self according to the genome DNA of the meloidogyne incognita,
the forward primer sequence MiF2:5'-CTACCCTTATCGGTGGATCACTA-3',
reverse primer MiR2: 5'-AATGACCCTGAACCAGACGTT-3';
TaqMan probe sequence MiP2:5'-AGTTTGACCTCAATGCGGCCATTTGCGT-3', the 5 'end of which is marked with fluorescein FAM group, and the 3' end is marked with TAMAR quenching group.
The results of comparing the primers of the present invention with 2 sets of primers with higher scores designed by software are shown in table 1 and fig. 1.
TABLE 1 qPCR detection results for the same template, different primers and probes
As shown in Table 1, under the same template and different primer conditions, the amplification Ct value of Mi-F2/R2 is smaller than that of other primers and the fluorescence signal value is higher, the fluorescence signals of Mi-F6/R6 and Mi-F7/R7 are both below 20, and the fluorescence signal value is lower, which indicates that the amplification cycle number corresponding to the fluorescence signal of the amplification product of Mi-F2/R2 reaching the set fluorescence threshold is small, and indicates that the amplification efficiency of the primer pair of the invention is higher than that of other primers.
The melting temperature (Tm) of the primer and the probe provided by the software is not optimal for real-time fluorescent quantitative PCR reaction, and the primer probe forms a stable secondary structure to reduce the utilization efficiency of the primer probe, so that the fluorescent signal is low, and the detection sensitivity is influenced. Therefore, the primer designed by software is not suitable for a TaqMan method, and the SYBR-Green I method is adopted for testing.
The results of comparing the primers of the present invention with the reported primers are shown in Table 2:
TABLE 2 qPCR detection results for the same template, different primers and probes
As shown in Table 2, the amplification Ct values of Mi-F2/R2 are smaller than those of other primers under the same template and different primer conditions, which indicates that the amplification cycle number corresponding to the fluorescence signal of the amplification product of Mi-F2/R2 reaching the set fluorescence threshold is small, and indicates that the amplification efficiency of the primer pair is higher than that of other primers.
Example 2 examination of the specificity of the primers of the invention
The invention uses oat heterodera avenae wollen, soybean heterodera glycines, Heterodera filipjevi, beet heterodera glycines, Stem nematode, Meloidogyne incognita, Brevibacterium sp.Stachys, Verticillium dahliae, Rhizoctonia solani AG-3, Rhizoctonia solani AG-4HGIII, Fusarium oxysporum wilt specialization FOV7, Fusarium oxysporum watermelon specialization Fon, Fusarium oxysporum cucumber specialization FOC1-2-11, colletotrichum gloeosporum as control, and ddH2And (4) O negative control. Extracting DNA of the nematode or soil-borne pathogenic bacteria, and carrying out PCR amplification by using a primer MiF2/MiR2, wherein the PCR conditions are as follows: rTaq 0.5. mu.L, MgCl2 3μL，10xPCR(Mg2+free)Buffer 2.5μL，dNTPs(25mm)2μL，MiF2(10μΜ)1μL，MiR2(10μΜ)，1μL，0.1％BSA5μL，MiP2 1μL，H2O4. mu.L, template 5. mu.L. PCR amplification procedure: 30s at 95 ℃; 94 ℃ for 5s, 60 ℃ for 30s, 45 cycles.
TABLE 3 qPCR assay with different DNA as template
Remarking: "-" indicates that no detection was made for the qPCR amplification using this DNA as a template.
The results are shown in table 3 and fig. 2, the primers of the present invention produced detection values by qPCR amplification using DNA of meloidogyne incognita as a template, while no detection values were produced by other insect DNAs as templates, indicating that the primers of the present invention have good specificity.
Example 3 examination of the sensitivity of primers
Carrying out PCR amplification by taking the specific primer Mi-F2/R2 of the meloidogyne incognita as a primer and the meloidogyne incognita genome as a template, cloning the amplification product to pMD18-T, and constructing a plasmid recombinant vector pMD 18-MiT. The extracted plasmid pMD18-MiT is cut by endonuclease EcoRI, the cut product is subjected to agarose Gel electrophoresis, then the corresponding fragment is recovered by cutting Gel, the fragment recovery is carried out by utilizing Gel Extraction Kit, the DNA concentration of the recombinant plasmid is measured by utilizing a Nanodrop 2000 spectrophotometer, and the copy number of the recombinant plasmid DNA is calculated according to a formula (Whelan et al, 2003).
By ddH2O gradient of the above plasmid vectorDiluting to 1 × 108、1×107、1×106、1×105、1×104、1×103、1×102、1×101、1×100Copies/. mu.L of dilutions.
The real-time fluorescent quantitative PCR reaction was carried out by using Taqman fluorescent probe method, the total volume was 20. mu.L, 2 XPerctStart TM II Pro2e qPCR SuperMix 10. mu.L, Mi-F2 (10. mu.M) 0.4. mu.L, Mi-R2 (10. mu.M) 0.4. mu.L, Mi-P2 (10. mu.M) 0.4. mu.L, and the copy number of the diluted DNA template was 5. mu.L. Each concentration gradient setup was repeated 3 times.
qPCR reaction procedure: the two-step method is adopted, the reaction comprises pre-denaturation at 94 ℃ for 1min, 1 cycle, and then 45cycles, wherein each cycle comprises 94 ℃ for 5s and 60 ℃ for 30 s. A Taqman probe detection technology is established by adopting a Roche LightCyclerL480 II quantitative detection system.
Copy number concentration (copies/. mu.L) ═ 6.02X 1023X concentration (ng/. mu.L). times.10-9) /(660 Xbase number).
The qPCR standard curve for detecting DNA of Meloidogyne incognita (Meloidogyne incognita) in the optimized reaction system is shown in FIG. 1, wherein the standard curve is-3.3336 x +41.057, R20.998, when the DNA concentration of Meloidogyne incognita is 1 × 101～1×108The linear relation is good in the copy/mu L range, and the method can be used for qPCR quantitative detection.
The lowest detectable value of each reaction system is 1 power of 10, namely 10 copies/. mu.L, and the template in the reaction system is 5. mu.L, so the sensitivity is 50 copies/reaction system. The sensitivity of the invention for detecting the DNA of the meloidogyne incognita is 50 copies/reaction system, and the invention is suitable for the quantitative detection of the DNA of the meloidogyne incognita.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Sequence listing
<110> institute of plant protection of academy of agriculture, forestry and science of Hebei province
<120> primer and probe for detecting meloidogyne incognita by real-time fluorescence quantitative PCR and application
<160> 3
<170> SIPOSequenceListing 1.0
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ctacccttat cggtggatca cta 23
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<213> Artificial Sequence (Artificial Sequence)
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aatgaccctg aaccagacgt t 21
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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agtttgacct caatgcggcc atttgcgt 28
Claims (10)
1. The primer and the probe for detecting the meloidogyne incognita by real-time fluorescent quantitative PCR are characterized in that the nucleotide sequence of the primer is as follows:
SEQ ID NO.1：5'-CTACCCTTATCGGTGGATCACTA-3'，
SEQ ID NO.2：5'-AATGACCCTGAACCAGACGTT-3'；
the nucleotide sequence of the probe is as follows:
SEQ ID NO.3：5'-AGTTTGACCTCAATGCGGCCATTTGCGT-3'。
2. the primer and probe for real-time fluorescence quantitative PCR detection of Meloidogyne incognita according to claim 1, wherein the 5 'end of the probe is labeled with a fluorescent group and the 3' end of the probe is labeled with a quencher group.
3. The primers and the probe for real-time fluorescence quantitative PCR detection of Meloidogyne incognita according to claim 1, wherein the 5 'end of the probe is labeled with fluorescein FAM group and the 3' end of the probe is labeled with TAMAR quencher group.
4. The use of the primers and probes for real-time fluorescent quantitative PCR detection of Meloidogyne incognita according to claim 1.
5. The application of the primer and the probe for detecting the meloidogyne incognita through the real-time fluorescence quantitative PCR as claimed in claim 1 in the aspect of detecting the meloidogyne incognita.
6. Kit for detecting meloidogyne incognita, comprising the primers and probes for real-time fluorescent quantitative PCR detection of meloidogyne incognita according to claim 1.
7. Use of the kit for detecting meloidogyne incognita according to claim 6.
8. The method for detecting the meloidogyne incognita by real-time fluorescence quantitative PCR, which is characterized by comprising the step of carrying out PCR amplification on DNA of the meloidogyne incognita by using the primer and the probe for detecting the meloidogyne incognita by real-time fluorescence quantitative PCR as claimed in claim 1.
9. The method for detecting meloidogyne incognita through real-time fluorescent quantitative PCR according to claim 8, wherein the PCR reaction system comprises the following components: rTaq 0.5. mu. L, MgCl23 μ L, 10XPCR Buffer 2.5 μ L, dNTPs 2 μ L, probe 1 μ L, primer 2 μ L, 0.1% BSA 5 μ L, H2O4. mu.L, template 5. mu.L.
10. The method for detecting meloidogyne incognita according to claim 8, wherein the PCR amplification procedure is: 30s at 95 ℃; 5s at 94 ℃; 60 ℃ for 30s, 45 cycles.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100081133A1 (en) * | 2006-07-10 | 2010-04-01 | Wageningen Universiteit | Methods of Detecting Root Knot Nematodes |
| CN106011246A (en) * | 2016-06-01 | 2016-10-12 | 宁波大学 | Isothermal amplification detection primer sequences for five meloidogyne spp. based on micro-fluidic chip technology and application of primer sequences |
-
2021
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100081133A1 (en) * | 2006-07-10 | 2010-04-01 | Wageningen Universiteit | Methods of Detecting Root Knot Nematodes |
| CN106011246A (en) * | 2016-06-01 | 2016-10-12 | 宁波大学 | Isothermal amplification detection primer sequences for five meloidogyne spp. based on micro-fluidic chip technology and application of primer sequences |
Non-Patent Citations (4)
| Title |
|---|
| KOKI TOYOTA ET,AL.: "Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita", 《SOIL SCIENCE AND PLANT NUTRITION》 * |
| 宋志强等: "土壤中南方根结线虫的实时荧光PCR检测和定量", 《植物保护学报》 * |
| 程燕等: "《生命科学实验仪器设备与使用》", 31 August 2014, 科学技术文献出版社 * |
| 邹克琴: "《基因工程原理和技术》", 31 January 2009, 浙江大学出版社 * |
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Application publication date: 20210907 |