CN106676193B - Molecular marker, primer and probe for identifying penicillium - Google Patents
Molecular marker, primer and probe for identifying penicillium Download PDFInfo
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- CN106676193B CN106676193B CN201710129262.1A CN201710129262A CN106676193B CN 106676193 B CN106676193 B CN 106676193B CN 201710129262 A CN201710129262 A CN 201710129262A CN 106676193 B CN106676193 B CN 106676193B
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Abstract
The invention discloses a molecular marker, a primer and a probe for identifying penicillium, wherein the ITS specific molecular marker of penicillium has a nucleotide sequence shown in SEQ ID NO. 1. The gene chip for specifically detecting the penicillium is designed aiming at the molecular marker, can carry out specific identification on the penicillium in a short time, improves the accuracy and the sensitivity of the penicillium identification, and has the characteristics of rapidness, accuracy and low cost.
Description
Technical Field
The invention belongs to the technical field of biological identification, relates to a method for identifying fungi, and particularly relates to an ITS specific molecular marker for identifying penicillium, and a special primer, a probe and a gene chip for the identification method.
Background
The strain of Penicillium,Penicillium(P.species) is a generic name for fungi of the genus Penicillium of the family Ascomycotina, Ascomycetales, Ascomycotina. The penicillium mycelium consists of a plurality of hyphae with transverse septa, and usually grows by producing conidia, and after the conidia fall off, the hyphae germinate in a proper environment. Penicillium species are many, and usually live on citrus fruits, vegetables, grains, and meatsAnd often on leather and food.
The penicillium is also a kind of mixed fungus which can cause pollution in the processes of edible fungus separation, strain production and cultivation, and can infect and harm in the processes of producing and cultivating edible fungi such as mushroom, oyster mushroom, shiitake mushroom, phoenix tail mushroom, straw mushroom, needle mushroom and the like, and is a common germ which influences the yield and quality of the edible fungi. Penicillium exists in various organic matters and is mainly saprophytic or weakly parasitic. Wheat bran, rice bran and other auxiliary materials in the culture material are breeding conditions of penicillium, and new conidia generated after diseases can be infected again through media such as artificial water spraying, air flow, insects and the like. After the penicillium invades the mushroom bed and pollutes the culture material, the growth of edible fungi hypha can be inhibited, so that the edible fungi hypha can not form fruit bodies, and even the fruit bodies are formed, the edible fungi can be browned and rotten. Therefore, the method has practical significance for rapidly detecting the penicillium in the edible fungi and the cultivation environment thereof.
For pathogenic bacteria which cannot be observed by naked eyes, if a conventional culture method is adopted, a detection period of at least 4-5 days is required, and the colony morphology of the pathogenic bacteria is not easy to clearly identify.
With the rapid development of molecular biology technology, especially the establishment and maturation of the technology divided into markers and gene markers, an effective means is provided for the development of simple, rapid and accurate fungal identification technology. The gene chip is a novel DNA recognition technology, and fungi can be effectively identified on the chip by utilizing the advantages of high flux and specificity of the chip so as to improve the detection accuracy. Meanwhile, the main operation steps of the gene chip detection are completed by instrument equipment, the detection period only needs 6-8 hours, the subjective experience of people in the traditional detection is not relied on, and the accurate detection and identification result can be obtained in a short time.
Disclosure of Invention
The invention aims to provide an ITS molecular marker for penicillium specificity so as to establish a rapid, sensitive and good-specificity penicillium identification method.
It is another object of the present invention to provide primers and nucleic acid probes for use in the method for identifying penicillium.
The invention adopts gene cloning technology and combines gene chip technology, firstly a section of highly conserved and highly specific nucleic acid sequence in the ITS DNA fragment of the penicillium is obtained to be used as a molecular marker for identifying the penicillium specifically; and then a group of specific primers and nucleic acid probes are designed and synthesized according to the molecular marker, a method for specifically identifying penicillium is established, and effectiveness evaluation tests are carried out on the established method.
In order to realize the aim, the nucleotide sequence of the penicillium ITS specific molecular marker is shown in SEQ ID NO. 1. The ITS specific molecular marker is a characteristic sequence which can be used as a gene chip detection target sequence and is determined by performing PCR amplification on total DNA extracted from penicillium by using a universal primer ITS1/ITS4, performing capillary sequencing on a PCR product, and performing full nucleic acid database comparison analysis on a sequencing result in NCBI.
Furthermore, according to the target sequence and according to the design principle of a primer and a probe, the invention designs a pair of primers which have the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 and are used for amplifying the ITS specific molecular marker, and a nucleic acid probe which has the nucleotide sequence shown in SEQ ID NO.4 and is used for detecting the ITS specific molecular marker.
More specifically, the present invention is to label a fluorescent reporter group Hex at the 5 'end of a primer 1 and subject the nucleic acid probe to an amination treatment, i.e., to link an amino group to the 5' end of the nucleic acid probe, and finally artificially synthesize a primer and a nucleic acid probe having the following nucleotide sequences.
Primer 1: 5 'Hex-GATCTTTCCTGAGTGAGGGCC-3'.
Primer 2: 5'-CTACAGAGCGGGTGACAAAGC-3' are provided.
Nucleic acid probe: 5' NH3-TTTTTTTTTTTTACACGGGTGGGGAGGTTGGACCCAGGA -3'。
The invention also provides a gene chip for detecting penicillium. The gene chip is prepared by adopting a conventional method in the field, and the nucleic acid probe is fixed on the gene chip. The film base adopted by the gene chip is preferably a conventional aldehyde film base so as to be matched with the aminated probe.
The invention also provides a kit for identifying penicillium, which at least comprises the gene chip or nucleic acid probe for detecting penicillium, a special primer and a PCR amplification reagent for amplifying the ITS specific molecular marker of the penicillium, and other necessary related reagents.
Finally, the invention provides a method for identifying penicillium, which identifies the penicillium by using the ITS specific molecular marker of the penicillium, and the amplification product of the penicillium obtained by using the PCR method contains a nucleotide sequence shown in SEQ ID NO. 1.
Specifically, the method for identifying penicillium comprises the following steps:
a) extracting the total genome DNA of a sample to be detected;
b) carrying out PCR amplification on the extracted total DNA by using the primers to obtain a PCR amplification product;
c) and carrying out in-situ hybridization on the PCR amplification product and the nucleic acid probe so as to specifically identify whether the sample to be detected contains penicillium.
After the PCR amplification product is hybridized with the nucleic acid probe in situ, the unhybridized PCR amplification product is washed away, and the hybridization result is detected. If the hybridization result is positive, the sample to be detected contains penicillium; and if the hybridization result is negative, the sample to be detected does not contain penicillium.
The invention preferably adopts a laser confocal scanner to carry out fluorescence scanning detection on the hybridization result.
In the above method of the present invention, the methods of extracting total genomic DNA, PCR amplification and in situ hybridization are also conventional.
The preferred PCR amplification procedure of the present invention is as follows: preheating at 95 ℃ for 5 min; 36 cycles: 95 ℃ for 20s, 58 ℃ for 20s, and 72 ℃ for 40 s; finally, extension is carried out for 5min at 72 ℃.
The special primer and the nucleic acid probe provided by the invention are used for identifying the penicillium and the edible fungi without the penicillium, and the result shows that only the PCR amplification product of the penicillium is hybridized and combined with the nucleic acid probe of the invention and shows a positive result (the detection site is lightened), and the PCR amplification products of other edible fungi or domestic fungi are not hybridized and combined with the nucleic acid probe of the invention (the detection site is not lightened), so that the primer designed by the invention can amplify the specific gene fragment of the penicillium and effectively combine with the nucleic acid probe of the penicillium. Meanwhile, different nucleic acid probes are adopted to hybridize with the PCR amplification product of the penicillium, only the nucleic acid probe of the invention has positive response, and other nucleic acid probes have no response, which shows that the specificity of the penicillium nucleic acid probe of the invention is better. The results prove that the detection method provided by the invention has good specificity and accuracy.
Therefore, the penicillium ITS specific molecular marker, the special primer and the nucleic acid probe provided by the invention can be applied to the rapid identification and detection of the endophytic fungi penicillium. The specific molecular marker identification method provided by the invention has a more accurate identification result than conventional morphological judgment, and has the advantages of short detection time and high accuracy compared with other detection methods, the detection time is only 8 hours, the traditional culture and chemical chromogenic reaction identification takes 10-15 days, and the antagonism test requires at least two weeks.
Drawings
FIG. 1 shows the results of the identification of different fungi by the nucleic acid probe of the invention.
In the figure, Cl is an alexandrium giganteum detection site, Hy is a smoke color pleuromum ostreatus detection site, L e is a phellinus L enteraria patoullardii detection site, Tr is a Tricholoma lepidorum detection site, Me is a Pleurotus striatus detection site, L y is a Lyophyllum decastes detection site, Pe is a penicillium detection site, and Cy is a cylindrica fungus detection site.
Detailed Description
The present invention is further described below with reference to specific examples, but it should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Various changes or modifications of the present invention based on the present invention should be made by those skilled in the art within the scope of the present invention.
The methods used in the following examples were carried out according to conventional methods and conditions or selected according to the commercial instructions unless otherwise specified. The primers, nucleic acid probes and sequence determination were synthesized and performed by Biotechnology engineering (Shanghai) Inc.
Example 1: extracting the genomic DNA of the penicillium.
500. mu.l of overnight-cultured penicillin bacterial solution (OD)280Value 0.6), freezing at-80 deg.C for 30min for full solidification; putting the solidified bacterium blocks into a grinder, fully grinding the bacterium blocks into powder in a liquid nitrogen environment, transferring the powder into 500 mu l CTAB, and carrying out water bath at 65 ℃ for about 30 min; cooling, adding 600 μ l phenol, chloroform and isoamyl alcohol (25: 24: 1), mixing, and centrifuging at 12000rpm for 15 min; the supernatant was aspirated into an EP tube, 600. mu.l of chloroform: isoamyl alcohol (24: 1) was added thereto, and centrifugation was carried out at 12000rpm for 15 min; sucking the supernatant, transferring the supernatant into a new centrifuge tube, adding 1/3 volumes of NaAc (3mol/l) and 1ml of anhydrous ethanol precooled at the temperature of between 20 and 20 ℃, uniformly mixing, and standing for 3 to 4 hours at the temperature of between 20 and 20 ℃; centrifuging at 12000rpm for 10min, discarding the supernatant, adding 75% ethanol into the centrifuge tube, washing for 2-3 times, placing on absorbent paper, and inverting and air drying; addition of ddH2O20. mu.l of the dissolved DNA was stored at-20 ℃.
Example 2: determination of ITS specific molecular markers.
The total DNA of Penicillium obtained in example 1 was used as a template, and the DNA was amplified by PCR using the universal primers ITS1/ITS4, and the PCR product was subjected to capillary sequencing to obtain detailed sequence information.
And (3) carrying out whole nucleic acid database comparison analysis on the sequencing result in NCBI, screening to obtain a fragment with high conservation, comparing and selecting the results of different selected sequences, and finally determining a characteristic sequence in the sequencing result, wherein the amino acid sequence of the specific gene fragment of the penicillium is shown as SEQ ID NO. 1.
After homology comparison and retrieval, the selected target sequence is determined to be a DNA sequence with higher specificity and can be used as a target sequence for gene chip detection.
Example 3: design of primers and nucleic acid probes.
According to the target sequence determined in example 2 as a molecular marker, the special primers shown in SEQ ID NO.2 and SEQ ID NO.3 and the nucleic acid probe shown in SEQ ID NO.4 were designed according to the principle of designing primers and nucleic acid probes.
Primer 1: 5 'Hex-GATCTTTCCTGAGTGAGGGCC-3'.
Primer 2: 5'-CTACAGAGCGGGTGACAAAGC-3' are provided.
Nucleic acid probe: 5' NH3-TTTTTTTTTTTTACACGGGTGGGGAGGTTGGACCCAGGA -3'。
Wherein, a fluorescent reporter group Hex is marked at the 5' end of the primer 1; the nucleic acid probe is aminated by linking an amino group to the 5' -end of the nucleic acid probe.
Example 4: and (3) preparing a gene chip.
The aminated nucleic acid probe in example 3 was spotted on an aldehyde-based plate at a certain concentration, left overnight at room temperature, eluted with eluent I (5 × SSC, 1% SDS) and eluent II (0.25 × SSC, 1% SDS) for 5min each, the probe which had not been immobilized was eluted, and then centrifuged to prepare a gene chip.
Example 5: PCR amplification and fluorescent labeling identification of the fungus to be detected.
The total DNA of the fungus is extracted by taking the fungus to be detected and using a SIGMA fungus genome extraction kit, and the extraction method and the steps are detailed in the specification.
The total DNA extracted above was amplified by PCR using the primer designed in example 3 and labeled with fluorescence, and the PCR amplification system was as follows.
The PCR amplification procedure was as follows: preheating at 95 ℃ for 5 min; 36 cycles: 95 ℃ for 20s, 58 ℃ for 20s, and 72 ℃ for 40 s; finally, extension is carried out for 5min at 72 ℃.
The PCR product obtained by amplification and the gene chip prepared in the embodiment 4 are subjected to in-situ hybridization, the mixture is kept at 42 ℃ for 40min, eluent I (5 × SSC, 1% SDS) and eluent II (0.25 × SSC, 1% SDS) are respectively used for eluting for 5min, the PCR amplification product which is not hybridized is washed away, a laser confocal scanner is used for detecting the hybridization result of the gene chip, the detection site of the nucleic acid probe shows green fluorescence, the hybridization result is proved to be positive, the fungus to be detected contains penicillium, the hybridization result is negative if the detection site of the nucleic acid probe has no fluorescent bright point, and the fungus to be detected does not contain penicillium.
Example 6: and (3) verifying the specificity of the nucleic acid probe to the penicillium.
To prove the specific response of the nucleic acid probe of the invention to penicillium, 6 common edible fungi without penicillium infection are selected: alexander clitocybe maxima, phoma fumosoroseum, phellinus igniarius, lepista clavata sharp mushroom, tricholoma giganteum, Lyophyllum decastes and another common endophyte cylindrospora which is contained in edible fungi, the Alexandrium clavatum extract total DNA together with penicillium and perform PCR amplification, the in-situ hybridization is performed by using the nucleic acid probe, and the detection result is shown in figure 1.
The results show that neither edible fungi nor staphylotrichum infected with penicillium are detected, and only the detection site of penicillium is developed, thus proving that the target sequence, the corresponding nucleic acid probe and the special primer designed by the invention can specifically detect penicillium.
Then, a plurality of different penicillium samples are collected to extract total DNA, the special primer is used for PCR amplification, the primers are hybridized with a nucleic acid probe in situ, and the detection result shows that the hybridization results of all penicillium samples are positive, thereby proving to have specificity.
Example 7: detection specificity of the nucleic acid probe.
In order to further verify the reliability and the resolution of the nucleic acid probe, another 7 different nucleic acid probes are selected and used for carrying out in-situ hybridization on the PCR amplification product of the total DNA of the penicillium together with the nucleic acid probe. Wherein the nucleic acid probe 1 is a nucleic acid probe used in the present invention.
Nucleic acid probe 1: 5' NH3-TTTTTTTTTTTTACACGGGTGGGGAGGTTGGACCCAGGA-3'。
Nucleic acid probe 2: 5' NH3-TTTTTTTTTTTTGAAAAGATAGACCAGAAATATAAGAGA-3'。
Nucleic acid probe 3: 5' NH3-TTTTTTTTTTTTACCTCGGAAAATAGAATCCAGGTCTA-3'。
Nucleic acid probe 4: 5' NH3-TTTTTTTTTTTTAAGTGTATATGGACAAAGGCGAGGGGCG-3'。
Nucleic acid probe 5: 5' NH3-TTTTTTTTTTTTAGGCGTGCACATACATGCTCCGAAGGAG-3'。
Nucleic acid probe 6: 5' NH3-TTTTTTTTTTTTCTCAAGGACTGAATTACATTCATTACA-3'。
Nucleic acid probe 7: 5' NH3-TTTTTTTTTTTTCAACCCCCACATCCAAACCTAACCAAAC-3'。
Nucleic acid probe 8: 5' NH3-TTTTTTTTTTTTGACGGCGGGCGCGCGGCTCCCGGAGGTG-3'。
The procedure of the identification test was the same as in example 5. The detection result shows that only the nucleic acid probe 1 shows positive indication to the penicillium and other nucleic acid probes show negative indication, which indicates that the nucleic acid probe has very high specificity to the penicillium and can distinguish the penicillium from other fungi.
SEQUENCE LISTING
Institute of edible fungi of academy of agricultural sciences of Shanxi province (110)
Molecular marker, primer and probe for identifying penicillium of < 120 >
〈160〉 4
〈170〉 Patentin version 3.2
〈210〉 1
〈211〉 546
〈212〉 DNA
Penicillium (P. species) strain (213)
〈400〉 1
GATCTTTCCT GAGTGAGGGC CCTTCCTGGG TCCAACCTCC CCACCCGTGT TTATTGTACC 60
TTGTTGCTTC GGTGCGCCCG CCTCACGGCC GCCGGGGGGC TTCTGCCCCC GGGCCCGCGC 120
CCACCGAAGA CACCATTGAA CTCTGTCTGA AGATTGCAGT CTGAGCATAA ACTAAATAAG 180
TTAAAACTTT CAACAACGGA TCTCTTGGTT CCGGCATCGA TGAAGAACGC AGCGAAATGC 240
GATAACTAAT GTGAATTGCA GAATTCAGTG AATCATCGAG TCTTTGAACG CACATTGCGC 300
CCCCTGGTAT TCCGGGGGGC ATGCCTGTCC GAGCGTCATT GCTGCCCTCA AGCACGGCTT 360
GTGTGTTGGG CTCCGTCCCC CCGGGGACGG GCCCGAAAGG CAGCGGCGGC ACCGAGTCCG 420
GTCCTCGAGC GTATGGGGCT TTGTCACCCG CTCTGTAGGC CCGGCCGGCG CCAGCCGACA 480
ACCCATCATC CTTTTCAGGT TGACCTCGGA TCAGGTAGGG ATACCCGCTG AACTTAAGCA 540
TATCAA 546
〈210〉 2
〈211〉 21
〈212〉 DNA
< 213 > forward primer
〈400〉 2
GATCTTTCCT GAGTGAGGGC C 21
〈210〉 3
〈211〉 21
〈212〉 DNA
< 213 > reverse primer
〈400〉 3
CTACAGAGCG GGTGACAAAG C 21
〈210〉 4
〈211〉 39
〈212〉 DNA
Probe (213)
〈400〉 4
TTTTTTTTTT TTACACGGGT GGGGAGGTTG GACCCAGGA 39
Claims (7)
1. The ITS specific molecular marker of penicillium has a nucleotide sequence shown in SEQ ID No. 1.
2. A pair of primers for amplifying the penicillium ITS specific molecular marker of claim 1, having the nucleotide sequences shown in SEQ ID No.2 and SEQ ID No. 3.
3. A nucleic acid probe for detecting the penicillium ITS specific molecular marker of claim 1, having the nucleotide sequence of SEQ ID No. 4.
4. A gene chip for detecting Penicillium, wherein the nucleic acid probe according to claim 3 is immobilized on the gene chip.
5. A kit for identifying penicillium, comprising the nucleic acid probe of claim 3 and the primer pair of claim 2.
6. A method for identifying penicillium, which comprises the step of identifying by using the ITS specific molecular marker of the penicillium as defined in claim 1, wherein an amplification product obtained by using a PCR method comprises a nucleotide sequence shown as SEQ ID NO. 1.
7. The method for identifying penicillium according to claim 6, comprising:
a) extracting the total genome DNA of a sample to be detected;
b) performing PCR amplification on the extracted total DNA by using the primer of claim 2 to obtain a PCR amplification product;
c) and (3) carrying out in-situ hybridization on the PCR amplification product and the nucleic acid probe of claim 3 to specifically identify whether the sample to be detected contains penicillium.
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Citations (2)
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|---|---|---|---|---|
| CN101705300A (en) * | 2009-11-05 | 2010-05-12 | 北京大学第一医院 | Special probe and gene chip used for identifying penicillium marneffei |
| CN103635592A (en) * | 2011-06-09 | 2014-03-12 | 东洋制罐集团控股株式会社 | Method for detecting fungi, reaction solution for PCR, and carrier for detecting fungi |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705300A (en) * | 2009-11-05 | 2010-05-12 | 北京大学第一医院 | Special probe and gene chip used for identifying penicillium marneffei |
| CN103635592A (en) * | 2011-06-09 | 2014-03-12 | 东洋制罐集团控股株式会社 | Method for detecting fungi, reaction solution for PCR, and carrier for detecting fungi |
Non-Patent Citations (3)
| Title |
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| 7 株青霉菌的分离与鉴定;迟归兵等;《农业灾害研究》;20140715;22-24 * |
| GenBank: JX140750.1;Visagie,C.M等;《NCBI-GenBank》;20160718;1 * |
| 利用ITS核酸序列分析鉴定海洋青霉菌;曲凌云等;《安徽农业科学》;20091110;103-104 * |
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Inventor after: Li Yanting Inventor after: Zhu Min Inventor after: Guo Shang Inventor after: Nan Xiaojie Inventor after: Qu Huiling Inventor after: Guo Xiaofei Inventor after: Wang Hua Inventor after: Zhou Lin Inventor before: Zhou Lin Inventor before: Guo Shang Inventor before: Wang Hua |
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