AU2021104367A4 - Specific primer for detecting sugarcane pokkah boeng pathogen and detection method - Google Patents
Specific primer for detecting sugarcane pokkah boeng pathogen and detection method Download PDFInfo
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- 240000000111 Saccharum officinarum Species 0.000 title claims abstract description 65
- 235000007201 Saccharum officinarum Nutrition 0.000 title claims abstract description 64
- 244000052769 pathogen Species 0.000 title abstract description 31
- 230000001717 pathogenic effect Effects 0.000 title abstract description 31
- 238000001514 detection method Methods 0.000 title abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 238000000137 annealing Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 abstract description 6
- 238000012360 testing method Methods 0.000 abstract 4
- 108020004414 DNA Proteins 0.000 description 18
- 241000196324 Embryophyta Species 0.000 description 8
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- 239000012895 dilution Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108020001027 Ribosomal DNA Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
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- 230000002538 fungal effect Effects 0.000 description 1
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- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008654 plant damage Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
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Classifications
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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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses a specific primer for detecting sugarcane pokkah boeng pathogen
and a detection method. The specific primers Fp-F3/Fp-R3 were designed according to the
rDNA-ITS sequence of the sugarcane pokkah boeng pathogen, the reaction system and
procedures were optimized, and the specific primers for the detection of sugarcane pokkah
boeng pathogen and its detection method were established. By using the specific primers and
method of the present invention, it is only necessary to determine whether the test sample is
infected by the sugarcane pokkah boeng pathogen by determining whether there is a specific
band in the electrophoresis result, and there is no need to sequence the amplified product to
confirm, which improves the specificity and accuracy of the test, saving testing time and
reducing testing costs.
Description
Specific primer for detecting sugarcane pokkah boeng pathogen and
detection method
The invention relates to a specific primer for detecting sugarcane pokkah boeng
pathogen and a detection method, belonging to the technical field of plant protection.
Pokkah boeng is an important worldwide fungal disease of sugarcane. According
to reports, the disease incidence in India is 51.4%, resulting in a 24.9% reduction in
sugarcane yield and a loss of 25 billion rupees per year. Pokkah boeng is distributed
in all sugarcane planting provinces in China, and the diseased plant rate of Guitang 11,
the main cultivated variety planted in Guangxi reached 52.4% in the 1980 s. Yuetang
57-423 and Yuetang 54-176 were highly susceptible to this disease in Guangdong in
1989. Since 2000, with the extensive promotion of susceptible varieties such as ROC
1, ROC 10, ROC 16, ROC 22 in sugarcane areas in China, sugarcane pokkah boeng
occurred frequently and became increasingly serious. Especially in recent years, rainy
and high humidity, coupled with the large-scale planting of susceptible varieties
Yuetang 93-159, ROC 25 and Chuantang 79-15, led to the outbreak of sugarcane
pokkah boeng in Yunnan, resulting in serious yield and sugar reduction. The
investigation results showed that the occurrence of susceptible varieties were
seriously, which often caused a large number of sugarcane stalks to die. The average
diseased plants rate was 81.1%, and in severe cases it reached 100%. The average
measured yield loss was 38.42%, and reached 48.5% in severe cases. Sugar content
decreased by 3.14% on average and 4.21% at most in severe cases, which greatly
affected the high-quality development of sugar industry. Planting and breeding
disease-resistant varieties based on pathogen research is the most economical and effective measure to control sugarcane pokkah boeng disease, so rapid and accurate identification of sugarcane pokkah boeng pathogen is of great significance for disease control.
At present, the observation and identification of sugarcane pokkah boeng in the
field is easily confused with sugarcane leaf scorch disease and drought sugarcane
plant injury, but the pathogen detection and identification are mostly confirmed by
indoor isolation and culture of strains, and then PCR detection and sequencing with
fungal universal primers, which are expensive, time-consuming and labor-consuming,
and the accuracy and credibility of the identification results also depend on the
technical level and experience of researchers to a large extent, which is difficult to
meet the requirements of accurate and rapid control of plant protection. In order to
control sugarcane pokkah boeng scientifically and effectively, and realize rapid and
accurate detection and identification of sugarcane pokkah boeng, it is urgent to
establish a rapid detection method for sugarcane pokkah boeng pathogen which can
achieve the goal of "convenience", "rapidity" and "accuracy". According to the
invention, specific primers Fp-F3/ Fp-R3 are designed based on the ribosomal DNA
non-internal transcribed spacer (rDNA-ITS) sequence of the sugarcane pokkah boeng
pathogen, and a PCR rapid detection method of the sugarcane pokkah boeng pathogen
is established by optimizing the reaction system and the annealing temperature, so
that the detection operation is simple, the accuracy is good, the sensitivity is high, and
the time consumption is short; and after the reaction is finished, whether the
sugarcane pokkah boeng pathogen exists in the sample can be judged according to the
position of the specific amplified fragment.
The invention provides a rapid, sensitive and specific detection method of
sugarcane pokkah boeng pathogen and a specific primer.
The invention provides a specific primer for detecting sugarcane pokkah boeng,
which consists of a primer Fp-F3 and a primer Fp-R3, wherein the nucleotide
sequence of the primer Fp-F3 is shown as SEQ ID NO:1, and the nucleotide sequence
of the primer Fp-R3 is shown as SEQ ID NO:2.
The method for detecting sugarcane pokkah boeng pathogen provided by the
invention comprises the following steps:
(1) Reaction system: the 25 pL PCR reaction system contains 2 pL of DNA
template, 12.5 pL of Taq PCRMaster Mix, 2.5 pL of primer Fp-F3 and 2.5 pL of
primer Fp-R3 (10 pmol/L), and 5.5 pL of sterilized ddH20. And the nucleotide
sequence of the primer Fp-F3 is shown in SEQ ID NO:1, and the nucleotide sequence
of the primer Fp-R3 is shown in SEQ ID NO:2;
(2) Reaction procedure: pre-denaturation at 95°C for 5 min, denaturation at 94°C
for 30 s, annealing at 63°C for 15 s, extension at 72°C for 30 s, 30 cycles; At last, it
extends for 10 min at 72°C.
(3) The PCR products were subjected to 1.5% agarose gel electrophoresis. If a
single band of 400 bp was amplified, the detected samples were infected with
sugarcane pokkah boeng, and if this band was not amplified, the detected samples did
not have sugarcane pokkah boeng.
The invention has the advantages and beneficial effects that:
According to the invention, specific primers are designed by using the rDNA
ITS sequence of the sugarcane pokkah boeng pathogen, and a method for detecting
the sugarcane pokkah boeng pathogen with high sensitivity, short time consumption
and low cost is established. Compare with that existing detection method, the primer and the method provided by the invention have high detection sensitivity, and the specific target band can still be amplified after the genome DNA of the pokkah boeng pathogen is diluted by 104, and the sugarcane pokkah boeng pathogen can be accurately detected even under the condition of slight infection. The genomic DNA of sugarcane pokkah boeng can amplify a single band of 400 bp, which has strong specificity, little interference from exogenous genomic DNA and high accuracy. In addition, the method has the characteristics of short time consumption, simple operation, good reproducibility and low detection cost, does not need complex detection places and laboratories, completes the whole detection procedure in about 2 hours, and can determine whether sugarcane is infected with pokkah boeng pathogen through whether the electrophoresis results have specific bands, and does not need to sequence the products, thus being simple, rapid and strong in specificity, saving detection time and reducing detection cost.
SEQ ID NO:1 in the sequence table shows the nucleotide sequence of primer Fp
F3 for detecting sugarcane pokkah boeng pathogen.
SEQ ID NO:2 in the sequence table shows the nucleotide sequence of primer Fp
R3 for detecting sugarcane pokkah boeng pathogen.
Figure 1: The PCR detection electrophoresis diagram of specific primer Fp
F3/Fp-R3 on sugarcane pokkah boeng pathogen, where M is DNA Marker B, lane 1-2:
leaf samples infected with sugarcane pokkah boeng pathogen, lane 3: Leaf samples
infected with the pathogen of sugarcane leaf scorch, lane 4: leaf samples infected with
the pathogen of sugarcane smut, lane 5: leaf samples of drought-affected sugarcane
plants, lane 6: leaf samples of healthy sugarcane, lane 7: ddH20.
Figure 2: Sensitivity detection electrophoresis diagram of the specific PCR
detection system for sugarcane pokkah boeng pathogen, where M is DNA Marker C,
Lane 1: template DNA concentration dilution gradient is 10; Lane 2: template DNA
concentration dilution gradient is 10-1, Lane 3: template DNA concentration dilution
gradient is 10-2, Lane 4: template DNA concentration dilution gradient is 10-, Lane 5:
template DNA concentration dilution gradient is 10-4, Lane 6: template DNA
concentration dilution gradient is 10-5 5. Lane 7: ddH20.
Specific embodiments of the present invention are described in detail below to
facilitate further understanding of the present invention, and conventional methods are
not specifically described in the embodiments.
1. Primer design
A specific primer was designed based on the ribosomal DNA non-internal
transcribed spacer (rDNA-ITS) sequence of sugarcane pokkah boeng pathogen, and
the expected amplification target fragment was 400 bp. The designed specific primer
for detecting sugarcane pokkah boeng pathogen consists of primer Fp-F3 and primer
Fp-R3. The nucleotide sequences of primer Fp-F3are shown in SEQ ID NO:1 and the
nucleotide sequences of primer Fp-R3are shown in SEQ ID NO:2.The specific primer
is sent to a biological company for synthesis.
2. Total DNA extraction
0.1 g of sugarcane leaves with typical symptoms of sugarcane pokkah boeng,
sugarcane leaf scorch and sugarcane smut, and leaves of drought-affected sugarcane
plants and healthy sugarcane were taken, and the total DNA of leaves was extracted
by using plant genomic DNA extraction kit (taking Easy Pure plant Genomic DNA
Kit of Beijing Perfect Gold Biotechnology Company as an example). The specific
steps were operated according to the instructions, and PCR amplification,
electrophoresis detection and result judgment were carried out according to the
following methods.
3. PCR amplification
The extracted DNA was amplified by PCR using specific primers Fp-F3/ Fp-R3
of sugarcane pokkah boeng. The 25 pL PCR reaction system contained 2 pL of DNA
template, 12.5 pL of Taq PCR Master Mix 12.5 pL of each primer Fp-F3 and Fp-R3
(10 pmol/L), and 5.5 pL of ddH20 was sterilized. The reaction procedure is: pre
denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 63°C for 15
s, extension at 72°C for 30 s, and 30 cycles. At last, it extends for 10 min at 72°C.
4. Electrophoretic detection and result discrimination
The 5 pL PCR products were detected by electrophoresis with 1.5% agarose gel.
as shown in fig. 1, a single band of about 400 bp was amplified in sugarcane samples
infected with sugarcane pokkah boeng, but no band was amplified in sugarcane leaf
scorch, sugarcane smut samples, drought-affected sugarcane plants, healthy sugarcane
and water control.
5. Sensitivity determination of PCR detection method
Template DNA of sugarcane pokkah boeng pathogen was diluted with sterilized
double distilled water for 10 times, and then amplified with specific primers Fp-F3/
Fp-R3. Results as shown in fig. 2, the template DNA of sugarcane pokkah boeng can
still amplify a specific band of 400 bp after dilution of 10 -4.
Claims (2)
1. A specific primer for detecting sugarcane pokkah boeng, which is
characterized by comprising a primer Fp-F3 and a primer Fp-R3, wherein the
nucleotide sequence of the primer Fp-F3 is shown in SEQ ID NO:1 and the nucleotide
sequence of the primer Fp-R3 is shown in SEQ ID NO:2.
2. The method for detecting sugarcane pokkah boeng by using the specific
primer of claim 1, characterized in that:
(1) Reaction system: the 25 pL PCR reaction system contains 2 pL of DNA
template, 12.5 pL of Taq PCR master mix, 2.5 pL of primer Fp-F3 and 2.5 pL of
primer Fp-R3 (10 pmol/L), and 5.5 pL of sterilized ddH20;
(2) Reaction procedure: pre-denaturation at 95°C for 5 min, denaturation at 94°C
for 30 s, annealing at 63°C for 15 s, extension at 72°C for 30 s, 30 cycles; At last, it
extends for 10 min at 72°C;
(3) The PCR products were subjected to 1.5% agarose gel electrophoresis; If a
single band of 400 bp was amplified, the detected samples were infected with
sugarcane pokkah boeng, and if this band was not amplified, the detected samples did
not have sugarcane pokkah boeng.
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| AU2021104367A AU2021104367A4 (en) | 2021-07-21 | 2021-07-21 | Specific primer for detecting sugarcane pokkah boeng pathogen and detection method |
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| Publication Number | Publication Date |
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| AU2021104367A4 true AU2021104367A4 (en) | 2021-09-16 |
Family
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| AU (1) | AU2021104367A4 (en) |
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2021
- 2021-07-21 AU AU2021104367A patent/AU2021104367A4/en not_active Ceased
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