A dual PCR method for rapid detection of Siamese anthracnose and fruit anthracnose

Abstract

The present invention discloses a specific primer set for detecting Siamese anthrax and fruit anthrax and a corresponding dual PCR method for rapid detection of the two anthrax. The sequences of the primer set are shown in SEQ ID NO.1 to SEQ ID NO.4. The method of the present invention can be widely used to detect Siamese anthrax from various host sources ( Colletotrichum siamense ) and Colletotrichum oleraceus ( Colletotrichum fructicola ), and the method does not need to use multi-gene phylogenetic analysis and pathogenicity determination methods for analysis and identification during the operation process, which greatly saves time, manpower and cost. In addition, the specific primer set of the present invention also has the characteristics of strong specificity, good stability and high sensitivity, which further enhances the application value of the present invention in detecting these two anthrax bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of plant pathogenic organism detection, and particularly relates to a double PCR method for rapidly detecting siam anthracnose and fruit anthracnose. Background Art

[0002] Anthrax Colletotrichum Corda ), also known as the genus Spiny Colletotrichum, is a genus of the family Hypocrea, in the phylum Ascomycetes, class Hypocrea, subclass Hypocrea, family Hypocrea. Glomerulus ) has a wide range of hosts, including vegetables, forage, fruit trees, flowers and other important economic crops. Field surveys have shown that Siamese anthracnose ( Colletotrichum siamense ) and Colletotrichum oleraceus ( Colletotrichum fructicola ), is the dominant species in the field that causes anthracnose in rubber trees, and the pathogens have similar morphology, making identification methods cumbersome. C. siamese and C. fructicola The identification method still relies on traditional PCR technology combined with multi-gene construction of phylogenetic tree identification. The above method can only identify one type of anthrax at a time, which is time-consuming and costly. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention compares the rubber tree anthracnose C. siamese and C. fruit-growing The specific fragments on the two anthrax genomes were found, and a specific primer was designed on each specific fragment. Finally, the specificity and sensitivity of the two pairs of primers were tested to establish a rapid detection method. C. siamese and C. fructicola Duplex PCR detection method.

[0004] The technical solution of the present invention mainly includes the following contents:

[0005] One of the purposes of the present invention is to provide a specific primer set for detecting Siamese anthrax and fruit anthrax, comprising a primer pair 1 and a primer pair 2, wherein the nucleotide sequence of the primer pair 1 is:

[0006] CS-F: 5'-GTGAGTACATTTACCACCA-3' (SEQ ID NO. 1);

[0007] CS-R: 5'-GACCACACATCTGGCCAT-3' (SEQ ID NO. 2);

[0008] The nucleotide sequence of the primer pair 2 is:

[0009] CF-F: 5'- GTGACCTTCTCCCCTGAT-3' (SEQ ID NO. 3);

[0010] CF-R: 5'-CTGTATTTGTCTCCCAAAGC-3' (SEQ ID NO. 4).

[0011] The second object of the present invention is to provide a PCR kit for detecting Siamese anthrax and fruit anthrax, which contains the specific primer set.

[0012] Furthermore, the kit also contains PCR premix and ddH2O.

[0013] The third object of the present invention is to provide the use of the specific primer set or the PCR kit in detecting Siamese anthrax and fruit anthrax.

[0014] The fourth object of the present invention is to provide a dual PCR method for rapid detection of Siamese anthrax and fruit anthrax, comprising the following steps: extracting DNA of the sample to be tested, performing PCR amplification using the specific primer set, and determining the result based on the size of the amplified product.

[0015] Furthermore, the method for determining the result is: if the size of the amplified product is 220bp, the sample to be tested contains Siamese anthrax; if the size of the amplified product is 448bp, the sample to be tested contains fruit anthrax; if the size of the amplified product is 448bp, the sample to be tested contains fruit anthrax; if amplified products of 220bp and 448bp appear at the same time, the sample to be tested contains Siamese anthrax and fruit anthrax.

[0016] Furthermore, the sample to be tested includes anthrax or a sample containing anthrax, and the anthrax includes Siamese anthrax and / or fruit anthrax.

[0017] Furthermore, each 25 μL of PCR amplification system contains: 1 μL of each sample DNA to be tested, 1 μL of each upstream and downstream primer of two pairs of primers, 12 μL of PCR premix, and the balance is ddH2O.

[0018] Furthermore, the PCR premix is ​​2×Es Taq MasterMix (Dye). 2×Es Taq MasterMix (Dye) is a commercially available PCR premix, comprising Taq DNA polymerase, Mg 2+ , dNTPs, PCR stabilizers and enhancers and other ingredients.

[0019] The PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s, for 30 cycles; and extension at 72°C for 5 min.

[0020] Beneficial effects of the present invention:

[0021] Compared with the prior art, the method of the present invention is more effective for rubber trees ( Brazilian rubber tree ),mango( Mangosteen ),litchi( Chinese lychee ),cucumber( Cucumber ) and chili pepper ( Red pepper ) and other host sources C. siamese and C. fructicola All of them can be quickly detected and the corresponding bands can be amplified, indicating that the primers are widely used in detecting plants. C. siamese and C. fructicola The method does not require multi-gene phylogenetic analysis and pathogenicity determination for analysis and identification, which greatly saves time, manpower and cost. In addition, the above detection primers also have the characteristics of strong specificity, good stability and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the specificity test results of the CS primer pair of the present invention for single PCR;

[0023] Figure 2 Schematic diagram of the results of a single PCR specificity test of the CF primers of the present invention;

[0024] Figure 3 This is a schematic diagram of the dual PCR system of the present invention detecting different DNA concentrations;

[0025] Figure 4 Schematic diagram of the double PCR system of the present invention for detecting anthrax from rubber trees;

[0026] Figure 5 Schematic diagram of the dual PCR system of the present invention for detecting anthrax bacteria from different sources. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0028] The upstream and downstream primer sequences of the CS primer pairs described in the following examples are:

[0029] CS-F: 5'-GTGAGTAATTTTACCACCA-3'

[0030] CS-R: 5'- GACCACACATCTGGCCAT-3'

[0031] The upstream and downstream primer sequences of the CF primer pairs described in the following examples are:

[0032] CF-F: 5'- GTGACCTTCTCCCCTGAT-3'

[0033] CF-R: 5'-CTGTATTTGTCTCCCAAAGC-3'

[0034] Example 1

[0035] This example is the specificity test result of single PCR with CS primer pair:

[0036] Extraction laboratory originally identified the rubber tree source C. siamese and other anthrax DNA templates, add CS primer pairs, construct a 25μL PCR system, and perform PCR amplification. The 25μL PCR system is: 1μL DNA template, 1μL upstream and downstream primers, 12μL 2×Es Taq MasterMix (Dye), and add ddH2O to 25μL. Reaction procedure: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 30s, amplification for 28 cycles; 72℃ final extension for 5min, and the amplified product is stored at 4℃. Take 5μL PCR product for detection by agarose gel electrophoresis.

[0037] The results are as follows Figure 1 As shown, lanes 1-3 in the figure are C.siamense (Siamese Anthrax), lanes 4-10 are C . fruit-growing (Anthracnose fungi), C . Asian (Asian Anthrax), C . riddle ( Hidden Anthrax), C . tropical (Tropical Anthrax), C . laticiferous (Lactobacillus anthracis), C . Wanningen (Wanning anthrax) and blank control. The amplified band size in lanes 1-3 was 220bp, and no bands appeared in other lanes, indicating that the primers had high specificity.

[0038] Example 2

[0039] This example is the specificity test result of single PCR of CF primer pair:

[0040] Extraction laboratory originally identified the rubber tree source C. fructicolaand other anthrax DNA templates, add CF primer pairs, construct a 25μL PCR system, and perform PCR amplification. The 25μL PCR system is: 1μL DNA template, 1μL upstream and downstream primers, 12μL 2×Es Taq MasterMix (Dye), and add ddH2O to 25μL. PCR reaction program: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 30s, amplification for 30 cycles; 72℃ final extension for 5min, and the amplified product is stored at 4℃. Take 5μL PCR product for agarose gel electrophoresis for detection.

[0041] The results are as follows Figure 2 As shown, lanes 1-3 in the figure are C. fructicola , Lanes 4-10 are C. siamese , C.asianum , C.aenigma , C.tropicale , C. laticiphilum , C.wanningense The amplified band size in lanes 1-3 was 448 bp, and no bands appeared in other lanes, indicating that the primers had high specificity.

[0042] Example 3

[0043] This example is a dual PCR system for detecting anthrax from rubber trees.

[0044] Extraction of the rubber tree anthracnose fungus previously identified in the laboratory C . Siamese and C . fruit-growing DNA template, C . Siamese and C . fruit-growing The DNA templates were combined with other anthrax DNAs, and CS and CF primer pairs were added to construct a 25μL PCR system for PCR amplification. The 25μL PCR system was as follows: 1μL of DNA template, 1μL of upstream and downstream primers of the two primer pairs, 12μL of 2×Es Taq MasterMix (Dye), and ddH2O was used to make up to 25μL. The reaction procedure was as follows: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 30s, and amplification for 30 cycles; 72℃ final extension for 5min, and the amplified product was stored at 4℃. Take 5μL of PCR product for detection by agarose gel electrophoresis.

[0045] The results are as follows Figure 3 As shown, lane 1 is C. siamese and C. fructicola , Lanes 2-6 are C.siamense and C . Asian , C . riddle , C . tropical , C . laticiferous , C . Wanningen Mix separately, lanes 7-11 are C . fruit-growing and C . Asian , C . riddle , C . tropical , C . laticiferous , C . Wanningen The results showed that when the DNA template had C.siamense When the DNA template has C . fruit-growing A 448bp band was also amplified when the DNA template was used, but no band appeared when using other DNA templates.

[0046] Example 4

[0047] This example is a sensitivity test of a dual PCR reaction system:

[0048] Detection by spectrophotometer C.siamense and C. fructicola The DNA concentration was 1 ng / µL, and then diluted tenfold. C.siamense and C. fructicola The DNA concentrations were 1 ng / µL, 0.1 ng / µL, 0.01 ng / µL, 0.001 ng / µL, and 0.0001 ng / µL, respectively, and the sensitivity of duplex PCR was determined.

[0049] Establishment of duplex PCR reaction system: C. siamese and C. fructicola 1µL of DNA template, 1μL of upstream and downstream primers of two pairs of primers, 12μL of 2×Es Taq MasterMix (Dye), and ddH2O to make up to 25μL. Reaction procedure: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 30s, amplification for 30 cycles; 72℃ final extension for 5min.

[0050] The results are as follows Figure 4As shown, the DNA template concentrations of lanes 1-5 are 1 ng / µL, 0.1 ng / µL, 0.01 ng / µL, 0.001 ng / µL, and 0.0001 ng / µL, respectively, and lane 6 is a blank control. The results show that when the DNA template concentration is greater than 0.01 ng / µL, detection can be performed, indicating that the dual PCR method established by the present invention has high sensitivity.

[0051] Example 5

[0052] This example is a dual PCR system for detecting anthrax from different host sources.

[0053] Extraction laboratory previously identified mango, lychee, cucumber and pepper sources C. siamese and C. fruit-growing DNA, CS and CF primer pairs were added, and the PCR reaction system and reaction procedure described in Example 4 were used for verification.

[0054] The results are as follows Figure 5 As shown, lane 1 is from mango C.siamense and C . fruit-growing Lane 2 is from litchi C . Siamese and C . fruit-growing Lane 3 is from cucumber C . fruit-growing Lane 4 is from pepper C . Siamese Lane 5 is a blank control. The results show that this specific primer can be widely used to detect C . Siamese and C . fruit-growing , when the DNA template has C . Siamese When the DNA template has C. fructicola A 448bp band could also be amplified.

[0055] Further explanation: In actual detection, the detection can be performed by directly extracting DNA from host plant samples infected with anthrax, and the host plants include rubber trees, mangoes, lychees, cucumbers, peppers, etc. Experiments have shown that plant tissue DNA has no interference with the detection of anthrax.

[0056] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of a specific primer set in detecting Siamese anthrax and fruit anthrax, characterized in that: The primer set includes primer pair 1 and primer pair 2; The nucleotide sequence of the primer pair 1 is: CS-F: 5'-GTGAGTAATTTTACCACCA-3' CS-R: 5'- GACCACACATCTGGCCAT-3' The nucleotide sequence of the primer pair 2 is: CF-F: 5'- GTGACCTTCTCCCCTGAT-3' CF-R: 5'-CTGTATTTGTCTCCCAAAGC-3'; The Siamese anthrax and fruit anthrax are derived from rubber trees, mangoes, cucumbers or peppers.

2. A dual PCR method for rapid detection of Siamese anthrax and fruit anthrax, characterized in that: The following steps are involved: Extract the DNA of the sample to be tested, perform PCR amplification using primer pair 1 and primer pair 2, and determine the result based on the size of the amplified product; The nucleotide sequence of the primer pair 1 is: CS-F: 5'-GTGAGTAATTTTACCACCA-3' CS-R: 5'- GACCACACATCTGGCCAT-3' The nucleotide sequence of the primer pair 2 is: CF-F: 5'- GTGACCTTCTCCCCTGAT-3' CF-R: 5'-CTGTATTTGTCTCCCAAAGC-3'; The Siamese anthrax and fruit anthrax are derived from rubber trees, mangoes, cucumbers or peppers.

3. The dual PCR method according to claim 2, characterized in that: The method for determining the result is: if the size of the amplified product is 220bp, the sample to be tested contains Siamese anthrax; if the size of the amplified product is 448bp, the sample to be tested contains fruit anthrax; if the size of the amplified product is 448bp, the sample to be tested contains fruit anthrax; if amplified products of 220bp and 448bp appear at the same time, the sample to be tested contains Siamese anthrax and fruit anthrax.

4. The dual PCR method according to claim 2, characterized in that: The sample to be tested includes anthrax bacteria or a sample containing anthrax bacteria, and the anthrax bacteria includes Siamese anthrax bacteria and / or fruit anthrax bacteria.

5. The dual PCR method according to claim 2, characterized in that Each 25 μL PCR amplification system contains: 1 μL of the sample DNA to be tested, 1 μL of the upstream and downstream primers of the two pairs of primers, 12 μL of PCR premix, and the balance is ddH2O.

6. The dual PCR method according to claim 5, characterized in that The PCR premix solution is 2×Es TaqMasterMix.

7. The dual PCR method according to claim 2, characterized in that The PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s, for 30 cycles; and extension at 72°C for 5 min.

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