CN112725494B - Method and primer for detecting 17 tomato pathogens by using micro-fluidic chip - Google Patents
Method and primer for detecting 17 tomato pathogens by using micro-fluidic chip Download PDFInfo
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Abstract
The invention discloses a method and primers for detecting 17 tomato pathogens by using a microfluidic chip. The invention provides a composition for identifying or assisting in identifying tomato disease pathogens, which can consist of 17 primer pairs in SEQ ID Nos. 1-34 in a sequence table. The composition for identifying or assisting in identifying the tomato disease pathogens is added into a microfluidic chip to carry out fluorescence PCR reaction, and the detection of 17 common tomato disease pathogens can be completed by reading fluorescence signals of PCR products. The sensitivity and specificity of the detection are high, and the rapid identification of tomato pathogens is facilitated.
Description
Technical Field
The invention belongs to the field of microbial molecule detection, and particularly relates to a method and primers for detecting 17 tomato pathogens by using a microfluidic chip.
Background
Tomatoes are the second only consumed from potatoes in the world. With the continuous expansion of the tomato cultivation area and the increase of the planting years, the development of diseases on the tomatoes tends to be more serious year by year. Tomato is reportedly infested with over 200 pathogens, including fungi, bacteria, viruses and nematodes. Diseases frequently occurring in tomatoes produced mainly include early blight caused by infection with Alternaria solani (As), leaf spot disease caused by infection with corynebacterium solani (corynebacterium cassifolia, Cc), gray mold caused by infection with Botrytis cinerea (Bc), root rot caused by infection with Phytophthora capsici (phythophoora capsici, Phc), Pythium aphanidermatum (Pythium anhydrium, Pa) and Fusarium solani (Fs), gray leaf spot caused by infection with Stemphylium solani (Sts), blight caused by infection with Fusarium oxysporum (Fo), blight caused by infection with Sclerotinia sclerotiorum (sclerotiorum sclerotium, sclerotium sorium), blight caused by infection with Rhizoctonia solani (sclerotiorum sorium, sclerotiorum), blight caused by infection with Rhizoctonia solani (sclerotiorum sorium, sclerotiorum), Rhizoctonia solani blight caused by infection with Rhizoctonia solani, sclerotiorum solani, Rhizoctonia solani (Rhizoctonia solani, Sclerotinia solani, etc.; bacterial spot disease caused by infection of Pseudomonas syringae tomato pathogenic variety (Pst), bacterial wilt caused by solanaceae Ralstonia solanacearum (Ras), bacterial canker caused by clausii microorganism subsp.microzyme, Cmm, soft rot disease caused by carrot soft rot Pectobacterium subsp.carotovorum, Pcc, and bacterial scab caused by Xanthomonas campestris pathogenic variety capsicum spot (Xanthomonas campestris pv. vesicaria, Xcv); tomato viral diseases caused by infection with Tomato Yellow Leaf Curl Virus (TYLCV) and the like. The pathogens can infect tomatoes, so that the tomato yield and quality are seriously affected, the normal production of the tomatoes is seriously hindered, and huge economic loss is caused to vast farmers. Because some pathogens cause symptoms on tomatoes with certain similarities, misdiagnosis of diseases is easily caused, and thus, improper control methods are caused. Therefore, it is important to accurately identify various pathogens on the tomato with the disease, especially to detect the pathogens at the early stage of the disease.
The traditional detection methods for plant pathogens such as microscopic examination and culture morphology identification are time-consuming and labor-consuming, have high requirements for professional knowledge of diagnosticians, and are not suitable for bacteria and viruses. The immunoassay technology can effectively identify pathogens, but has the defects of low sensitivity and easy generation of false positive. Molecular biology techniques developed in recent years are increasingly used to detect pathogens on plants. Such as classical PCR, nested PCR, fluorescent quantitative PCR, random amplified polymorphic DNA marker, isothermal amplification, etc., have been used for the detection of phytopathogenic fungi, bacteria and viruses. At present, molecular biological detection technology is established for various pathogens on tomatoes, the detection of a single sample can be realized within 4 hours, and the accuracy and the sensitivity basically meet the actual requirements. However, the above method can only identify one or a few pathogens at the same time, and other pathogens on tomato plants are easily missed. Multiplex PCR can detect multiple targets in one reaction tube, but typically no more than 6 targets can be detected at one time. Because the phenomena of frequent diseases of tomato plants in fields and complex occurrence of various diseases are more, a method for simultaneously detecting various pathogens is urgently needed.
Microfluidic chips (also known as Miniaturized Total Analysis Systems, TAS) or lab-on-a-chip (lab) are designed to achieve the overall miniaturization, automation, integration and portability from sample processing to detection by the cross-over of analytical chemistry, micro-electro-mechanical Systems (MEMS), computers, electronics, materials, and biology and medicine. The microfluidic chip analysis system has the characteristics of high analysis speed, high sensitivity, high selectivity, miniaturization and the like, and common detection methods mainly comprise electrochemical detection, mass spectrometry detection, optical detection and the like. At present, microfluidic chips have been widely used in gene detection, protein and amino acid analysis, cell analysis, immunoassay, single molecule detection, drug analysis and screening, enzyme analysis, and the like. In the field of microbial detection, microfluidic chip detection technology has been used for detection of human beings, aquatic organisms, and food-related fields. In the aspect of plant pathogen detection, researchers have applied the plant pathogenic nematode to detection, but reports of application of the plant pathogenic nematode to detection of other plant pathogenic microorganisms such as fungi, bacteria, viruses and the like are not found.
Disclosure of Invention
The invention aims to solve the technical problem of how to simultaneously detect 17 common tomato-infecting pathogens or how to improve the detection efficiency of tomato-infecting pathogens.
In order to solve the above technical problems, the present invention provides, in the first place, a composition for identifying or assisting in identifying a pathogen of tomato disease.
The composition consists of n primer pairs in the following 17 primer pairs, wherein n can be any natural number from 17 to 1:
A1) a primer pair with the number of N23_1A, which consists of two single-stranded DNA molecules shown in SEQ ID No.1 and SEQ ID No. 2; or a primer pair consisting of 22 th to 42 th nucleotides of SEQ ID No.1 and a single-stranded DNA molecule shown in SEQ ID No. 2;
A2) a primer pair with the number of N23_2A consisting of two single-stranded DNAs shown as SEQ ID No.3 and SEQ ID No. 4; or a primer pair consisting of 22 th to 41 th nucleotides of SEQ ID No.3 and a single-stranded DNA molecule shown in SEQ ID No. 4;
A3) a primer pair with the number of N23_3A consisting of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No. 6; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.5 and a single-stranded DNA molecule shown in SEQ ID No. 6;
A4) a primer pair with the number of N23_4A consisting of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No. 8; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.7 and a single-stranded DNA molecule shown in SEQ ID No. 8;
A5) a primer pair with the number of N23_5B consisting of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No. 10; or a primer pair consisting of 22 th to 47 th nucleotides of SEQ ID No.9 and a single-stranded DNA molecule shown in SEQ ID No. 10;
A6) a primer pair with the number of N23_6B consisting of two single-stranded DNAs shown as SEQ ID No.11 and SEQ ID No. 12; or a primer pair consisting of 22 th to 47 th nucleotides of SEQ ID No.11 and a single-stranded DNA molecule shown in SEQ ID No. 12;
A7) a primer pair with the number of N23-7D consisting of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No. 14; or a primer pair consisting of 22 nd to 43 th nucleotides of SEQ ID No.13 and a single-stranded DNA molecule shown in SEQ ID No. 14;
A8) a primer pair with the number of N23_8B consisting of two single-stranded DNAs shown as SEQ ID No.15 and SEQ ID No. 16; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.15 and a single-stranded DNA molecule shown in SEQ ID No. 16;
A9) a primer pair with the number of N23_9A consisting of two single-stranded DNAs shown in SEQ ID No.17 and SEQ ID No. 18; or a primer pair consisting of 22 th to 48 th nucleotides of SEQ ID No.17 and a single-stranded DNA molecule shown in SEQ ID No. 18;
A10) a primer pair with the number of N23-10D consisting of two single-stranded DNAs shown as SEQ ID No.19 and SEQ ID No. 20; or a primer pair consisting of 22 th to 43 th nucleotides of SEQ ID No.19 and a single-stranded DNA molecule shown in SEQ ID No. 20;
A11) a primer pair with the number of N23_11A consisting of two single-stranded DNAs shown as SEQ ID No.21 and SEQ ID No. 22; or a primer pair consisting of 22 nd to 44 th nucleotides of SEQ ID No.21 and a single-stranded DNA molecule shown in SEQ ID No. 22;
A12) a primer pair with the number of N23_12C consisting of two single-stranded DNAs shown as SEQ ID No.23 and SEQ ID No. 24; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.23 and a single-stranded DNA molecule shown in SEQ ID No. 24;
A13) a primer pair with the number of N23_13E consisting of two single-stranded DNAs shown as SEQ ID No.25 and SEQ ID No. 26; or a primer pair consisting of 22 th to 45 th nucleotides of SEQ ID No.25 and a single-stranded DNA molecule shown in SEQ ID No. 26;
A14) a primer pair with the number of N23_14A consisting of two single-stranded DNAs shown as SEQ ID No.27 and SEQ ID No. 28; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.27 and a single-stranded DNA molecule shown in SEQ ID No. 28;
A15) a primer pair with the number of N23-15B consisting of two single-stranded DNAs shown as SEQ ID No.29 and SEQ ID No. 30; or a primer pair consisting of 22 nd to 47 th nucleotides of SEQ ID No.29 and a single-stranded DNA molecule shown in SEQ ID No. 30;
A16) a primer pair with the number of N23_16A consisting of two single-stranded DNAs shown as SEQ ID No.31 and SEQ ID No. 32; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.31 and a single-stranded DNA molecule shown in SEQ ID No. 32;
A17) a primer pair with the number of N23-17B consisting of two single-stranded DNAs shown as SEQ ID No.33 and SEQ ID No. 34; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.33 and a single-stranded DNA molecule shown in SEQ ID No. 34.
The pathogen can be Pseudomonas syringae tomato pathogenic variant, Klebsiella mulleriensis subspecies, Ralstonia solanacearum, Pectinophyllum carotovorum subspecies, Xanthomonas campestris pepper spot pathogenic variant, Alternaria solani, Psychotria polystachyos, Phytophthora capsici, Botrytis cinerea, Stachybotrys solani, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum, Pythium melongena, Pseudocercosporium coalustum and/or tomato yellow leaf curl virus.
In order to solve the technical problems, the invention also provides a method for detecting the tomato disease pathogen, which comprises the following steps:
B1) adding the above-mentioned N23_1A, N23_2A, N23_3A, N23_4A, N23_5B, N23_6B, N23_7D, N23_8B, N23_9A, N23_10D, N23_11A, N23_12C, N23_13E, N23_14A, N23_15B, N23_16A and/or N23_17B primer pairs into the chip reaction area of the microfluidic chip respectively;
B2) adding the solution of the PCR reaction system except the primers into the microfluidic chip in B1) through the sample inlet hole;
B3) placing the microfluidic chip obtained in B2) into a PCR instrument for fluorescence PCR reaction;
B4) the chip obtains a fluorescence signal by scanning, and whether the pathogen exists is determined according to the fluorescence signal value (or a fluorescence signal scanning map).
The pathogen can be Pseudomonas syringae tomato pathogenic variant, Klebsiella mulleriensis subspecies, Ralstonia solanacearum, Pectinophyllum carotovorum subspecies, Xanthomonas campestris pepper spot pathogenic variant, Alternaria solani, Psychotria polystachyos, Phytophthora capsici, Botrytis cinerea, Stachybotrys solani, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum, Pythium melongena, Pseudocercosporium coalustum and/or tomato yellow leaf curl virus.
The microfluidic chip described in B1) above may be a PCR reaction plate comprising 4 parallel reaction zones. Each reaction zone comprises 1 sample inlet hole, 1 sample outlet hole, 1 sample channel, 28 reaction cells and 28 connecting channels. The volume of the PCR reaction pool may be 1. mu.L.
The PCR reaction may be a gradient PCR reaction. The gradient PCR reaction procedure may be: 15min at 95 ℃; 20s at 95 ℃ and 60s at 61 ℃; 20s at 95 ℃ and 60s at 60.6 ℃; 20s at 95 ℃ and 60s at 60.2 ℃; 20s at 95 ℃ and 60s at 59.8 ℃; 20s at 95 ℃ and 60s at 59.4 ℃; 20s at 95 ℃ and 60s at 59 ℃; 20s at 95 ℃ and 60s at 58.6 ℃; 20s at 95 ℃ and 60s at 58.2 ℃; 20s at 95 ℃ and 60s at 57.8 ℃; 20s at 95 ℃ and 60s at 57.4 ℃; 20s at 95 ℃ and 60s at 57 ℃; 26 cycles as follows: 20s at 95 ℃ and 60s at 57 ℃; finally 10 ℃ for 60 s.
In order to solve the technical problems, the invention also provides a product for identifying or assisting in identifying the pathogen of the tomato disease.
The product may be a reagent or a kit or system. The reagent or kit contains the composition for identifying or assisting in identifying the pathogen of tomato disease as described above. The system may also contain the composition described above and/or the microfluidic chip in the method described above.
Any of the following uses of the compositions described above also fall within the scope of the present invention:
p1, the use of said composition for identifying or aiding in the identification of a tomato pathogen;
p2 and the application of the composition in identifying or assisting in identifying tomato diseases.
Any of the following uses of the above described method also fall within the scope of the invention:
p3, the use of the method for identifying or assisting in identifying tomato pathogens;
p4, and the application of the method in identifying or assisting in identifying tomato diseases.
Any of the following uses of the products described above also fall within the scope of the invention:
p5, the use of said product for identifying or aiding in the identification of tomato pathogens;
p6, and the application of the product in identifying or assisting in identifying tomato diseases.
Compared with the prior art, the invention has the advantages that:
1. the method can simultaneously identify pseudomonas syringae tomato pathogenic variety, clavibacter michiganensis subspecies, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic variety, alternaria solani, polyspora capsici, phytophthora capsici, botrytis cinerea, stemphylium solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphanidermatum, pseudotailia solanacearum and tomato yellow leaf curl virus at one time. The target gene sequences of five pathogens, namely, the clavibacter michiganensis subspecies, the phomopsis solani, the fusarium solani, the rhizoctonia solani and the sclerotinia sclerotiorum, for designing specific amplification primers are developed for the first time and are used for specifically detecting five pathogens.
2. The number of disposable detection samples is large: the method can detect 17 pathogens at one time; meanwhile, one chip can detect 4 samples and/or 112 targets at one time.
3. The sample dosage is less: the volume of each reaction cell is 1.0 mu L, namely, each detection index is only 1 mu L of reaction liquid, so that the dosage of reaction reagents can be obviously reduced.
3. The specificity is strong: the primer sequence of each tomato pathogen is designed according to the conserved region of the target gene of each species, and BLAST comparison verification and fluorescence PCR verification prove that the primer sequence has strong specificity and can obviously distinguish the pathogens on the tomato.
The primer pair composition provided by the invention is used for detecting 17 tomato pathogens, and the accurate detection of various tomato pathogens can be realized by carrying out fluorescence PCR reaction on a microfluidic chip. The detection specificity is strong, and 17 pathogens can be specifically detected; the detection sensitivity is high, and the volume of a reaction system of each sample is less than 1 mu L; the detection efficiency is high, 17 tomato pathogenic pathogens can be simultaneously identified at one time, 4 samples and/or 112 targets can be simultaneously detected at one time by one chip, and the rapid identification of the tomato pathogens is facilitated.
Drawings
Fig. 1 is a schematic design diagram of a microfluidic chip used in the present invention.
Fig. 2 is a schematic diagram of a microfluidic chip used in the present invention.
FIG. 3 is a scanned graph of the specificity verification result of 17 pairs of primers specific to tomato pathogens in the present invention.
FIG. 4 is a scanned graph of the field-collected sample detection results using the microfluidic fluorescent PCR system established in the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 establishment of method for detecting tomato pathogens by fluorescence PCR
1. Designing a primer: 1 pair of primers are respectively designed according to the whole genome sequences of gap1 of pseudomonas syringae tomato pathogenic variety, gyrB of clavibacter michiganensis, 16s rRNA of ralstonia solanacearum, species-specific probe sequence of pectobacterium carotovorum carrot subspecies, xanthomonas campestris pepper spot pathogenic variety, the whole genome of alternaria solani, TUB of corynebacterium polystachyos, beta tubulin of phytophthora capsici, TUB A of botrytis cinerea, GAPDH of stemphylium solani, RPB2 of fusarium solani, beta tubulin of fusarium oxysporum, RMK1 of rhizoctonia solani, RPB2 of sclerotinia sclerotiorum, COI of pythium aphanidermatum, Avr4 of pseudocercosporus solani and tomato yellow leaf curl virus, meanwhile, a section of universal fluorescent label sequence is added at the 5' end of the forward primer, a microfluidic chip fluorescent PCR detection system is established, and the detection of the 17 tomato pathogens is completed. The primer sequences of the 17 tomato pathogens are designed according to conserved regions of target genes of respective species, and are verified by BLAST comparison, so that the primers have specificity and can obviously distinguish the 17 pathogens. Wherein the content of the first and second substances,
the sequence of the specific primer pair (N23_1A) for detecting the pseudomonas syringae tomato pathogenic variety is as follows:
N23_1A-F1：5′-GAAGGTGACCAAGTTCATGCTCGACACTGTACACGGCCCATT-3' (SEQ ID No.1 of the sequence Listing);
The sequence of the specific primer pair (N23_2A) for detecting the Clavibacter michiganensis subspecies is:
N23_2A-F1：5′-GAAGGTGACCAAGTTCATGCTCGCGATGCAGTGGACCACCT-3' (SEQ ID No.3 of the sequence Listing);
n23_ 2A-R: 5'-GGTGTGGACGCTCTCCGTGTA-3' (SEQ ID No.4 of the sequence Listing);
the sequence of a specific primer pair (N23_3A) for detecting the Ralstonia solanacearum is as follows:
N23_3A-F1：5′-GAAGGTGACCAAGTTCATGCTCACGTCATACAATGGTGCATACAGA-3' (SEQ ID NO in sequence Listing)o.5)；
the sequence of a specific primer pair (N23_4A) for detecting the pectobacterium carotovorum subspecies is as follows:
N23_4A-F1：5′-GAAGGTGACCAAGTTCATGCTGATCAGTGGTACGACATCGTGAAAT-3' (SEQ ID No.7 of the sequence Listing);
the sequence of a specific primer pair (N23_5B) for detecting the Xanthomonas campestris pepper spot pathogenic variety is as follows:
N23-5B-F：5′-GAAGGTGACCAAGTTCATGCTATCGGAGTTTCTCTATCTTCTTATCA-3' (SEQ ID No.9 of the sequence Listing);
N23-5B-R: 5'-CGAGTATCTATCAGGTCAAGTTGTTG-3' (SEQ ID No.10 of the sequence Listing);
the sequence of a specific primer pair (N23_6B) for detecting the alternaria solani is as follows:
N23-6B-F：5′-GAAGGTGACCAAGTTCATGCTAGGCAAAAATTTACCTCATAAAGACA-3' (SEQ ID No.11 of the sequence Listing);
N23-6B-R: 5'-TGTATGTTCTTCCTTATGGGCAAA-3' (SEQ ID No.12 of the sequence Listing);
the sequence of the specific primer pair (N23-7D) used for detecting the Bacteroides polyspora is:
N23-7D-F：5′-GAAGGTGACCAAGTTCATGCTGCGACACGAACTAACTGGGACT-3' (SEQ ID No.13 of the sequence Listing);
N23-7D-R: 5'-TGAAGTAGACGTTCATGCGCTC-3' (SEQ ID No.14 of the sequence Listing);
the sequence of a specific primer pair (N23_8B) for detecting phytophthora capsici is as follows:
N23-8B-F：5′-GAAGGTGACCAAGTTCATGCTGGATAACGAAGCCCTGTACGATATT-3' (SEQ ID No.15 in the sequence Listing);
N23-8B-R: 5'-TCAGGTCACCGTAAGTGGGAGTAGT-3' (SEQ ID No.16 of the sequence Listing);
the sequence of a specific primer pair (N23_9A) for detecting the botrytis cinerea is as follows:
N23_9A-F：5′-GAAGGTGACCAAGTTCATGCTGATAGTTTGCCTGTGATATGGTTAGTT-3' (SEQ ID No.17 in the sequence Listing);
n23_ 9A-R: 5'-CTGCTTCGCGACAACCTCAGATT-3' (SEQ ID No.18 of the sequence Listing)
The sequence of the specific primer pair (N23_10D) for detecting the photinaca solani is as follows:
N23-10D-F：5′-GAAGGTGACCAAGTTCATGCTGCCATTTGTTGCTAGTGGATTG-3' (SEQ ID No.19 of the sequence Listing)
N23-10D-R: 5'-CGACCTTGATCTCACCCTTGAAC-3' (SEQ ID No.20 of the sequence Listing);
the sequence of a specific primer pair (N23_11A) for detecting the fusarium solani is as follows:
N23_11A-F：5′-GAAGGTGACCAAGTTCATGCTGTCAATGCCGACAACGTCAAGTA-3' (SEQ ID No.21 of the sequence Listing);
the sequence of the specific primer pair (N23_12C) for detecting Fusarium oxysporum is:
N23-12C-F：5′-GAAGGTGACCAAGTTCATGCTGTGCCTTTCCCTCGTCTACACTT-3' (SEQ ID No.23 of the sequence Listing);
N23-12C-R: 5'-GGTCAGAGGAGCAAAGCCAAC-3' (SEQ ID No.24 of the sequence Listing);
the sequence of a specific primer pair (N23_13E) for detecting the rhizoctonia solani is as follows:
N23-13E-F：5′-GAAGGTGACCAAGTTCATGCTCCATTTTGGACATCATTAAACCTC-3' (SEQ ID No.25 in the sequence Listing);
N23-13E-R: 5'-ACAGTACACCTCTTTGAACGCATC-3' (SEQ ID No.26 of the sequence Listing);
the sequence of a specific primer pair (N23_14A) for detecting sclerotinia sclerotiorum is as follows:
N23_14A-F：5′-GAAGGTGACCAAGTTCATGCTCTTGACATGAGCTTTTGTACGTTGA-3' (SEQ ID No.27 of the sequence Listing);
the sequence of a specific primer pair (N23_15B) for detecting the pythium aphanidermatum is as follows:
N23-15B-F：5′-GAAGGTGACCAAGTTCATGCTACCTGAATGTGCTTGTACGCTAGATA-3' (SEQ ID No.29 of the sequence Listing);
N23-15B-R: 5'-GTTGAATCTGGAGCAGGTACTGGT-3' (SEQ ID No.30 of the sequence Listing);
the sequence of a specific primer pair (N23_16A) for detecting the cercospora coalignis is as follows:
N23_16A-F：5′-GAAGGTGACCAAGTTCATGCTGTGTGATTGGCCTGAGAACACGA-3' (SEQ ID No.31 of the sequence Listing);
the sequence of a specific primer pair (N23_17B) for detecting the tomato yellow leaf curl virus is as follows:
N23-17B-F：5′-GAAGGTGACCAAGTTCATGCTAAGTCCAGTCTTATGAGCAACGG-3' (SEQ ID No.33 of the sequence Listing);
N23-17B-R: 5'-TCACTAACACAACGAACAATACCAG-3' (SEQ ID No.34 of the sequence Listing);
the base sequences underlined in the 17 pairs of primers are universal fluorescent tag sequences.
2. DNA extraction
The extraction of the fungal DNA adopts a CTAB method: inoculating fungal mycelia growing on PDA culture medium to PD liquid culture medium, performing shake culture at 28 deg.C and 180rpm for 10 days, and filtering the culture medium with gauze to collect mycelia. Weighing 0.2g of mycelium in a sterilized mortar, adding liquid nitrogen, quickly grinding into powder and transferring into a 1.5mL centrifuge tube; adding 700mL of 2 xCTAB solution, and carrying out water bath at 65 ℃ for 30 min; centrifuging at 12000rpm for 12min, collecting supernatant 600mL, adding 600mL chloroform-isoamyl alcohol (24:1) into a new 1.5mL centrifuge tube, and centrifuging at 12000rpm for 12 min; taking 400mL of the supernatant solution, adding 800mL of absolute ethyl alcohol into a new 1.5mL centrifuge tube, standing for 10min, and centrifuging at 12000rpm for 12 min; completely sucking the supernatant, adding 1mL of 75% ethanol, rinsing the precipitate, and centrifuging at 12000rpm for 5 min; completely sucking 1mL of 75% ethanol rinsing solution, naturally drying the precipitate, adding 50 μ L of dd H2O; the extracted DNA was quantified by a spectrophotometer and diluted to 10 ng/. mu.L and stored at-20 ℃.
The bacterial DNA extraction method comprises the following steps: the bacteria stored in the NA test tube were inoculated onto NB liquid medium, cultured with shaking at 28 ℃ and 160rpm for 18 hours, and then DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit (Ltd.: Tiangen Biochemical technology Co., Ltd.; Cat. No.: DP302), and the extracted DNA was quantitatively determined by a spectrophotometer and then diluted to 10 ng/. mu.L and stored at-20 ℃.
The plant or virus DNA extraction method comprises the following steps: weighing 1g of tomato leaf infected with tomato yellow leaf curl virus or healthy tomato leaf, extracting total DNA of the plant leaf and the virus according to the operation instruction of a plant genome DNA extraction kit (company: Tiangen Biochemical technology Co., Ltd., product number: DP305), and diluting the extracted DNA to 10 ng/. mu.L after the quantification by a spectrophotometer and storing the diluted DNA at-20 ℃.
3. Design and pointing system of micro-fluidic chip
The microfluidic chip provided by the invention is from Beijing Boao biotechnology, Inc. The chip contains 4 parallel reaction zones. Each reaction zone contains 1 well, 1 sample channel and 28 reaction cells and 28 connecting channels (fig. 1 and 2). Therefore, the chip of 4 parallel reaction zone contains a total of 112 reaction pools, if all used can simultaneously detect 112 targets. Before the chip is spotted, the corresponding relation between the reaction pool and each primer pair is determined. Then, the specific primer of each pathogen is spotted in the corresponding reaction tank, and after drying and sealing, the reaction tank is stored at 4 ℃ for standby. The volume of each primer was 0.14. mu.L (concentration 2. mu. mol/L).
4. Preparing a reaction system: the total volume of the fluorescent PCR reaction system is 40. mu.L, wherein 2 × Master PCR Mix (containing Taq polymerase, MgCl2dNTP, ROX, fluorescent marker sequence and a sequence containing a fluorescence Quencher (Quencher) at the 3' end complementary to the fluorescent marker sequence 20. mu.L, DNA 30ng, dd H2O was supplemented to 40. mu.L. The total volume of each parallel reaction zone was 40. mu.L.
5. Fluorescent PCR reaction on the microfluidic chip: the reaction system is added into the chip at one time through the sample inlet hole, and is centrifuged for 2min at 4000rpm, so that the mixed solution is uniformly dispersed into each reaction tank, and the volume of each reaction tank is about 1 mu L. The chip was then placed on a plate PCR apparatus FP4 (Boao Bio Inc.) to perform a fluorescent PCR reaction (single PCR reaction in each reaction well). The reaction program is 95 ℃ for 15min, 1 cycle; 20s at 95 ℃,20 s at 61-57 ℃ (gradient PCR) for 20s, 10 cycles (annealing temperature decreases in 10 cycles at-0.4 ℃/cycle: decrease by 0.4 ℃ in each cycle); 20s at 95 ℃, 60s at 57 ℃ and 26 cycles; finally 10 ℃ for 60s, 1 cycle. That is, the reaction sequence was (table 1): 15min at 95 ℃; 20s at 95 ℃ and 60s at 61 ℃; 20s at 95 ℃ and 60s at 60.6 ℃; 20s at 95 ℃ and 60s at 60.2 ℃; 20s at 95 ℃ and 60s at 59.8 ℃; 20s at 95 ℃ and 60s at 59.4 ℃; 20s at 95 ℃ and 60s at 59 ℃; 20s at 95 ℃ and 60s at 58.6 ℃; 20s at 95 ℃ and 60s at 58.2 ℃; 20s at 95 ℃ and 60s at 57.8 ℃; 20s at 95 ℃ and 60s at 57.4 ℃; 20s at 95 ℃ and 60s at 57 ℃; 26 cycles as follows: 20s at 95 ℃ and 60s at 57 ℃; finally 10 ℃ for 60 s.
TABLE 1 fluorescent PCR reaction procedure
6. Chip scanning: after the fluorescent PCR reaction is finished, the microfluidic chip is placed into a chip scanner Luxscan10K/D (Boo biological group, Inc.) for scanning, the result is processed by using Luxscan 3.0 software, and the data signal value is converted by the software. The scanning image has obvious red fluorescence signals, and the signals are positive if the signal value is more than or equal to 2500; otherwise, the result is negative. The final result is represented in the form of a scan.
Example 2 verification of specificity of tomato pathogen primers Using the microfluidic chip detection System designed by the present invention
Tomato pathogens (the pathogens Pseudocercosporella solanacearum, Fusarium niveum, Alternaria solani, Phytophthora infestans, Rhizoctonia solani, Botrytis cinerea, Phytophora torulosa, Phytophthora solani, Phytophthora capsici, Fusarium solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Pseudomonas syringae, Pseudomonas solani var. michiganensis subsp, Klebsiella solanacearum, Xanthomonas campestris, and pathogenic pepper spots were respectively extracted by the method established in example 1Varieties, carrot pectobacterium carotovorum subspecies, tomato yellow leaf curl virus, pythium aphanidermatum, verticillium dahliae and chicory pseudomonas can all be obtained from the group of vegetable disease comprehensive control subjects of vegetable and flower institute of academy of agricultural sciences, wherein pseudocercosporium coalignis, fusarium oxysporum, alternaria solani, phytophthora infestans, rhizoctonia solani, botrytis cinerea, polyspora cinerea, stemphylium solani, phytophthora capsici, fusarium solani, fusarium oxysporum and sclerotinia have been found in the literature "kanghua army, sai-sai, lichen, jiaguan, plum blossom. establishment and application of real-time fluorescent quantitative PCR detection technology for Pseudocercospora solani smus, plant protection report, 2019, 46 (6): 1214, 1221. "; pseudomonas syringae tomato pathogenic variants, Klebsiella mulleriensis subspecies, Ralstonia solanacearum, Xanthomonas campestris pepper spot pathogenic variants, pectobacterium carotovorum carrot subspecies, and Pseudomonas cichorii have been reported in the literature "Kanghuajun, Chailali, Cabernet Sauvignon, Schering, Guogu Jiang, Li Bao Ju. A quadruple PCR detection method for bacterial spot pathogen, ulcer pathogen, Ralstonia solanacearum and scab pathogen of tomato. 2254 and 2264; the tomato yellow leaf curl virus is disclosed in the literature "Chenlida, Caberelina, Xiechen, Caojinqiang, Chailali, Li Bao Ju. molecular detection and analysis of main tomato virus disease types in different areas of China. North China agricultural science, 2020,35 (1): 185-193. Pythium aphanidermatum and verticillium dahliae have been found in the literature "Liuruichi, Chengpo, Chailali, Dianaria, Xiechen, Paiguli, Li Bao Jue.establishment and application of triple PCR detection system for soil-borne pathogens of vegetables. 2069 and 2078. ) And the DNA of the healthy tissue of the tomato (Zhongza 9, Zhongshu vegetable science and technology (Beijing) Limited company), respectively using the designed primers of pseudomonas syringae tomato pathogenic variety, rod bacillus michiganensis subspecies michiganensis, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic variety, alternaria solani, polyspora, phytophthora capsici, botrytis cinerea, phomopsis solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphanidermatum, pseudotailed smus, tomato yellow kojiThe PCR reaction was carried out by using genomic DNAs of leaf virus, Pseudomonas cichorii (Psc), Phytophthora infestans (Pi), Verticillium dahliae (Vd) and Fusarium fulva (Ff) as reaction templates in accordance with steps 3, 4, 5 and 6 of the above example 1, and the specificity of 17 to the detection primer was verified by using dd H2O is blank control and healthy tomato plant tissue DNA is negative control. Wherein, the cichorium intybus, the phytophthora infestans, the verticillium dahliae and the brown mold are 4 non-target pathogens outside 17 detection target pathogens. The results are shown in (a) and (B) of fig. 3, fluorescent PCR amplification is performed on 17 target strain DNAs and 4 non-target strains by using 17 pairs of specific primers, each pair of primers can generate a fluorescent signal from the corresponding target strain DNA, but does not generate a fluorescent signal in the non-target strain DNA, which indicates that the designed primers and the established microfluidic chip fluorescent PCR method have good specificity.
Example 3 detection of tomato disease samples collected in the field by the microfluidic chip fluorescent PCR method established in the invention
18 disease samples (table 2) are collected from tomato producing areas (Wenzhou in Zhejiang, Hebei Heshui and Shandong Jinan) of different provinces in China, wherein 10 parts of leaf disease samples, 4 parts of root samples, 1 part of fruit samples and 3 parts of stem disease samples are collected. Carrying out isolation culture and common PCR identification on samples suspected of fungal and bacterial diseases, and determining the types of pathogens in the samples (table 3); the disease sample suspected of being a virus disease is identified by common PCR, and the virus type on the disease sample is determined (Table 3). The microfluidic chip fluorescence PCR reaction was performed according to steps 3, 4, 5, and 6 of example 1 above.
TABLE 2 sample Collection information
Table 3 detection results of tomato disease samples in different areas by different detection methods
Note: "/" indicates that no isolation culture was performed or no pathogen was isolated; "-" no pathogen detected;
the results show that tomato pathogens exist in 15 field samples through isolated culture and PCR identification, and no pathogens are detected in 3 field samples. The microfluidic chip provided by the invention is used for detecting field samples, and the detection result is consistent with the results of isolated culture and common PCR detection (table 3 and figure 4).
In conclusion, the detection method of the tomato pathogenic agents provided by the invention can simultaneously identify 17 tomato pathogenic agents at one time; 4 samples and/or 112 targets can be detected simultaneously and at one time by one chip; the detection sensitivity is high, and the volume of a reaction system of each sample is less than 1 mu L; the specificity is strong, and 17 pathogens can be specifically detected. By using the method disclosed by the invention, the accurate detection of various tomato pathogens can be realized, and further the rapid and accurate diagnosis of tomato diseases can be realized.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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Claims (9)
1. Composition for identifying or assisting in identifying pathogens of tomato diseases, characterized in that: the composition consists of the following 17 primer pairs:
A1) a primer pair with the number of N23_1A, which consists of two single-stranded DNA molecules shown in SEQ ID No.1 and SEQ ID No. 2; or a primer pair consisting of 22 th to 42 th nucleotides of SEQ ID No.1 and a single-stranded DNA molecule shown in SEQ ID No. 2;
A2) a primer pair with the number of N23_2A consisting of two single-stranded DNAs shown as SEQ ID No.3 and SEQ ID No. 4; or a primer pair consisting of 22 th to 41 th nucleotides of SEQ ID No.3 and a single-stranded DNA molecule shown in SEQ ID No. 4;
A3) a primer pair with the number of N23_3A consisting of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No. 6; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.5 and a single-stranded DNA molecule shown in SEQ ID No. 6;
A4) a primer pair with the number of N23_4A consisting of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No. 8; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.7 and a single-stranded DNA molecule shown in SEQ ID No. 8;
A5) a primer pair with the number of N23_5B consisting of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No. 10; or a primer pair consisting of 22 th to 47 th nucleotides of SEQ ID No.9 and a single-stranded DNA molecule shown in SEQ ID No. 10;
A6) a primer pair with the number of N23_6B consisting of two single-stranded DNAs shown as SEQ ID No.11 and SEQ ID No. 12; or a primer pair consisting of 22 th to 47 th nucleotides of SEQ ID No.11 and a single-stranded DNA molecule shown in SEQ ID No. 12;
A7) a primer pair with the number of N23-7D consisting of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No. 14; or a primer pair consisting of 22 th to 43 th nucleotides of SEQ ID No.13 and a single-stranded DNA molecule shown in SEQ ID No. 14;
A8) a primer pair with the number of N23_8B consisting of two single-stranded DNAs shown as SEQ ID No.15 and SEQ ID No. 16; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.15 and a single-stranded DNA molecule shown in SEQ ID No. 16;
A9) a primer pair with the number of N23_9A consisting of two single-stranded DNAs shown as SEQ ID No.17 and SEQ ID No. 18; or a primer pair consisting of 22 th to 48 th nucleotides of SEQ ID No.17 and a single-stranded DNA molecule shown in SEQ ID No. 18;
A10) a primer pair with the number of N23-10D consisting of two single-stranded DNAs shown as SEQ ID No.19 and SEQ ID No. 20; or a primer pair consisting of 22 th to 43 th nucleotides of SEQ ID No.19 and a single-stranded DNA molecule shown in SEQ ID No. 20;
A11) a primer pair with the number of N23_11A consisting of two single-stranded DNAs shown as SEQ ID No.21 and SEQ ID No. 22; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.21 and a single-stranded DNA molecule shown in SEQ ID No. 22;
A12) a primer pair with the number of N23_12C consisting of two single-stranded DNAs shown as SEQ ID No.23 and SEQ ID No. 24; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.23 and a single-stranded DNA molecule shown in SEQ ID No. 24;
A13) a primer pair with the number of N23_13E consisting of two single-stranded DNAs shown as SEQ ID No.25 and SEQ ID No. 26; or a primer pair consisting of 22 th to 45 th nucleotides of SEQ ID No.25 and a single-stranded DNA molecule shown in SEQ ID No. 26;
A14) a primer pair with the number of N23_14A consisting of two single-stranded DNAs shown as SEQ ID No.27 and SEQ ID No. 28; or a primer pair consisting of 22 th to 46 th nucleotides of SEQ ID No.27 and a single-stranded DNA molecule shown in SEQ ID No. 28;
A15) a primer pair with the number of N23_15B consisting of two single-stranded DNAs shown as SEQ ID No.29 and SEQ ID No. 30; or a primer pair consisting of 22 th to 47 th nucleotides of SEQ ID No.29 and a single-stranded DNA molecule shown in SEQ ID No. 30;
A16) a primer pair with the number of N23_16A consisting of two single-stranded DNAs shown as SEQ ID No.31 and SEQ ID No. 32; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.31 and a single-stranded DNA molecule shown in SEQ ID No. 32;
A17) a primer pair with the number of N23-17B consisting of two single-stranded DNAs shown as SEQ ID No.33 and SEQ ID No. 34; or a primer pair consisting of 22 th to 44 th nucleotides of SEQ ID No.33 and a single-stranded DNA molecule shown in SEQ ID No. 34;
the pathogens are pseudomonas syringae tomato pathogenic varieties, clavibacter michiganensis subspecies, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic varieties, alternaria solani, polyspora, phytophthora capsici, botrytis cinerea, photinia solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphani, rhizoctonia solani and tomato yellow leaf curl virus.
2. The method for detecting tomato disease pathogens comprises the following steps:
B1) adding the primer pairs N23_1A, N23_2A, N23_3A, N23_4A, N23_5B, N23_6B, N23_7D, N23_8B, N23_9A, N23_10D, N23_11A, N23_12C, N23_13E, N23_14A, N23_15B, N23_16A and N23_17B of claim 1 into the chip reaction area of the microfluidic chip respectively;
B2) adding the solution of the PCR reaction system except the primers into the microfluidic chip in B1) through the sample inlet hole;
B3) placing the microfluidic chip obtained in B2) into a PCR instrument for fluorescence PCR reaction;
B4) scanning the chip result to obtain a fluorescence signal, and determining whether a pathogen exists according to the fluorescence signal value;
the pathogens are pseudomonas syringae tomato pathogenic varieties, clavibacter michiganensis subspecies, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic varieties, alternaria solani, polyspora, phytophthora capsici, botrytis cinerea, photinia solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphani, rhizoctonia solani and tomato yellow leaf curl virus.
3. The method of claim 2, wherein: B1) the microfluidic chip is a PCR reaction flat plate comprising 4 parallel reaction areas; each reaction zone comprises 1 sample inlet hole, 1 sample outlet hole, 1 sample channel, 28 reaction cells and 28 connecting channels; the volume of the PCR reaction pool is 1 mu L.
4. A method according to claim 2 or 3, characterized in that: the PCR reaction is a gradient PCR reaction.
5. The method of claim 4, wherein: the gradient PCR reaction procedure was: 15min at 95 ℃; 20s at 95 ℃ and 60s at 61 ℃; 20s at 95 ℃ and 60s at 60.6 ℃; 20s at 95 ℃ and 60s at 60.2 ℃; 20s at 95 ℃ and 60s at 59.8 ℃; 20s at 95 ℃ and 60s at 59.4 ℃; 20s at 95 ℃ and 60s at 59 ℃; 20s at 95 ℃ and 60s at 58.6 ℃; 20s at 95 ℃ and 60s at 58.2 ℃; 20s at 95 ℃ and 60s at 57.8 ℃; 20s at 95 ℃ and 60s at 57.4 ℃; 20s at 95 ℃ and 60s at 57 ℃; 26 cycles as follows: 20s at 95 ℃ and 60s at 57 ℃; finally 10 ℃ for 60 s.
6. The product for identifying or assisting in identifying tomato disease pathogens is characterized in that: the product is a reagent or a kit or a system; the agent or kit or system comprising the composition for identifying or aiding in the identification of a pathogen of tomato disease as claimed in claim 1.
7. Use of the composition of claim 1 for any of the following:
use of P1, the composition of claim 1, for identifying or aiding in the identification of a tomato pathogen;
the use of P2, the composition of claim 1, for identifying or aiding in identifying tomato diseases;
the pathogens are pseudomonas syringae tomato pathogenic varieties, clavibacter michiganensis subspecies, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic varieties, alternaria solani, polyspora polystachya, phytophthora capsici, botrytis cinerea, photinia solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphani, rhizoctonia solani and tomato yellow leaf curl virus;
the disease is caused by the pathogen.
8. Use of the method of any one of claims 2 to 5 for any one of the following:
use of P3 or the method of any one of claims 2 to 5 for identifying or aiding in the identification of a tomato pathogen;
use of P4, the method of any one of claims 2 to 5, for identifying or aiding in identifying tomato diseases;
the pathogens are pseudomonas syringae tomato pathogenic varieties, clavibacter michiganensis subspecies, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic varieties, alternaria solani, polyspora polystachya, phytophthora capsici, botrytis cinerea, photinia solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphani, rhizoctonia solani and tomato yellow leaf curl virus;
the disease is caused by the pathogen.
9. Use of the product of claim 6 in any of the following applications:
use of P5, the product of claim 6, for identifying or aiding in the identification of tomato pathogens;
the use of P6, the product of claim 6, for identifying or aiding in identifying tomato diseases;
the pathogens are pseudomonas syringae tomato pathogenic varieties, clavibacter michiganensis subspecies, ralstonia solanacearum, pectobacterium carotovorum subspecies, xanthomonas campestris pepper spot pathogenic varieties, alternaria solani, polyspora polystachya, phytophthora capsici, botrytis cinerea, photinia solani, fusarium oxysporum, rhizoctonia solani, sclerotinia sclerotiorum, pythium aphani, rhizoctonia solani and tomato yellow leaf curl virus;
the disease is caused by the pathogen.
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