CN101419169B - Method for identifying bentgrass nematode by real time fluorescent PCR technology - Google Patents

Abstract

The invention discloses a method for identifying Anguina agrostis by applying a real-time fluorescent PCR technology. The specific DNA sequence of the Anguina agrostis is utilized; a bioinformatics method is used to design a specific primer and a specific probe; a 5' end of the probe is labeled with a FAM; a 3' end of the probe is labeled with TAMRA; a target gene of the Anguina agrostis is amplified by applying a fluorescent PCR method; and whether the Anguina agrostis is contained in a sample is identified through the change of a fluorescent signal of the probe during the reaction. The method is not affected by larva instar and the quantity of polypides, is quicker and more accurate compared with morphology and morphometry identification methods, has simple operation, can be standardized, solves the problem of a standard method for quickly identifying the Anguina agrostis, greatly saves detection time, accelerates the acceptance of grass seeds at ports, promotes the import and export trade, and saves the cost generated by the long-term storage of goods in a goods yard.

Description

A method for identification of bentgrass nematodes using real-time fluorescent PCR technology

technical field

The invention relates to a biological identification technology, in particular to a nematode molecular biological identification technology, that is, the technology of using real-time fluorescent PCR reaction to identify the nematode nematodes, especially the nematode nematodes bentgrass.

Background technique

Anguina agrostis is a species in the order Tylenchida, the suborder Tylenchina, the superfamily Tylenchoidea, the family Anguinidae, and the genus Anguina Important plant nematodes are important pests of pastures. Its host plants mainly include Agrostisspp., Calamagrostis spp., Carex spp., Dactylis spp., and Eragrostis spp. , Festuca spp., Hordeum spp., Lolium spp., Phleum spp., Poa spp., Puccinellias pp. .), Sporobolus spp., Trisetum spp., etc., mainly damage the inflorescence of the host plant, resulting in a decrease in the yield of seeds and shoots of the diseased plant. Especially in areas where forage such as bentgrass is widely planted, it poses a serious threat to production. In addition, bentgrain nematodes are also mediators of Lasseria bacillus, and they synergistically form yellow galls on the host caryopsis, which are highly toxic to animals, and can cause neurological disorders in cattle, horses, sheep and other animals, and animals eat them by mistake. After being poisoned, he even fainted, and finally died of convulsions.

After the risk analysis of Chinese experts, the clipgrain nematode is considered to be a very serious nematode, and it is listed as a plant quarantine nematode imported into China. In 1997, the State Bureau of Animal and Plant Quarantine issued the "Notice on the Implementation of Quarantine Measures for Clenteritis Nematodes", requiring all entry and exit ports in my country to strengthen the examination and approval and quarantine of imported plant seeds. Since then, my country's Shenzhen, Guangzhou, Tianjin and other ports have repeatedly intercepted bentgrass nematodes from imported grass seeds, and returned the entire batch of goods, which has aroused great concern from domestic and foreign traders and inspection and quarantine departments. Animal and plant quarantine agencies in the United States and other countries have asked exporters to avoid sending grass seeds containing the bentgrass nematode to China.

The identification of bentgrain nematodes is an important task in my country's entry-exit inspection and quarantine. However, there is no standard method for the identification of bentgrain nematodes in my country. The traditional morphological and morphometric identification methods are mainly based on the morphology of adult nematodes. It takes a long time to identify the biological characteristics and shape measurement data; and only when adults are found, combined with factors such as host plants and distribution areas, can be accurately identified. larvae, making accurate identification of bentgrass nematodes very difficult. Although the PCR amplification sequencing method studied in China and the PCR-RFLP method and multiplex PCR method studied in this laboratory can also accurately identify the nematode cnitting nematodes, the above two methods must have PCR amplification, gel electrophoresis and The procedure of gel imaging is complex and the identification time is long.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to solve the problem of the standard method for rapid identification of C. cinematodes and provide a method for using real-time fluorescent PCR technology to identify C. cinematodes.

The technical scheme of the present invention is to provide a kind of method utilizing real-time fluorescent PCR technology to identify the nematode bentgrass, comprising the following steps:

(1) Design and synthesize TaqMan probe sequences, and prepare standard plasmids;

(2) preparing the DNA template of the sample to be tested;

(3) Fluorescence PCR detection is carried out on the DNA template of the sample to be tested;

(4) Perform fluorescent PCR detection on the standard plasmid, and carry out positive control with the result of step (3) to obtain the detection result.

The design and synthesis of TaqMan probe sequence described in step (1) includes the following steps:

A, prepare the DNA template of standard product;

B. Perform PCR amplification, recover the amplified product, sequence, design and synthesize TaqMan probes according to the amplified gene sequence and primer sequence.

The PCR amplification conditions described in step B are: (1) 94° C., 3 minutes; (2) 30 cycles, wherein each cycle includes three steps of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 30 seconds; ( 3) 5 minutes at 72°C.

The amplified gene sequence described in step B is shown in SEQ ID NO: 1, namely

gtttgcctac cggttgttta cggccgtctt atcatgtctt ggctattgtagacgtatctg atggctgttt tcacaccgac tgcatgtgg

The primer sequence described in step B is:

Primer sequence-5'-CCACATGCAGTCGGTGTGAA-3;

Primer sequence two 5'-GTTTGCCTACCGGTTGTTTACG-3'.

The probe sequence described in step B is:

5'FAM-TCATGTCTTGGCTATTGTAGACGTATCTGA-TAMRA3'.

In the PCR reaction system of the PCR detection described in step (3) or step (4), the composition ratio of each component is as follows:

Composition Concentration Sample Amount

DNA sample 1μL;

PCR buffer 10 times 1μL;

MgCl2 25mM 0.5μL;

dNTP 10mM 0.2 μL;

Primer sequence 1 20μmol/L 0.2μL;

Primer sequence 2 20μmol/L 0.2μL;

Probe sequence 20μmol/L 0.1μL;

Taq enzyme 5U/μl 0.1μL;

Double distilled water 6.7μL;

Total volume 10μL;

Wherein the DNA sample is a standard plasmid or a DNA template of a sample to be tested.

The PCR detection amplification procedure described in step (3) or step (4) is:

(1) 94°C for 3 minutes;

(2) 94°C for 10 seconds;

(3) 30 seconds at 60°C;

(4) Go back to procedure (2) and repeat 45 times.

Using real-time probe fluorescent PCR technology (TaqMan probe method) to identify M. benthamiana includes: using this method to identify M. benthamiana, the sequence of primers, probes and amplified products, and the matching fluorescent PCR Reaction system and fluorescent PCR amplification conditions.

In the present invention, after step (1) designs two primer sequences and a probe sequence specific to C. benthamiana and the corresponding fluorescent PCR reaction conditions, in the process of using the probe in the reaction process It is only necessary to identify whether the sample contains C. nematodes c. nematodes by the change of the fluorescent signal in the sample, and it is not necessary to repeat the processes of PCR amplification, gel electrophoresis and gel imaging for each detection.

Compared with the prior art, the technical advantage of the present invention is to be able to improve the speed of detection and sensitivity and specificity, and its beneficial effect is: (1) provide a kind of fast real-time detection method of St. The blank of real-time fluorescent PCR technique identification bentgrass nematode has the technical basis of establishing relevant detection standardization; (2) the implementation of the present invention can accelerate the quarantine speed of the port inspection and quarantine agency to import and export grass seeds, accelerate the port inspection and release speed, reduce The time and cost of storing goods in the warehouse; can improve the inspection level of our port inspection and quarantine agencies, improve the status and image of our inspection and quarantine agencies in international trade; can promote foreign businessmen and quarantine agencies to strengthen the export of grass seeds to China Nematode quarantine work to avoid the entry of grass seeds containing bentgrass nematodes into China.

Description of drawings

Fig.1 Galls intercepted from bentgrass seeds (Bent grass) imported from Oregon, USA

Figure 2 Galls of Anguina tritici preserved in our laboratory in the 1970s

Figure 3 Galls intercepted from teff seeds imported from South Africa on May 20, 2001

Figure 4 Amplification curve

Figure 5 Amplification curve

Detailed ways

(1) Design and synthesize TaqMan probe sequences and prepare standard plasmids

Nematode materials Anguina agrostis comes from galls intercepted from bentgrass seeds (Bent grass) imported from Oregon, USA in December 2000. The galls are cigar-shaped, dark brown, and about normal in size 2 times the size of the seeds, see Figure 1, a large number of live larvae were isolated from this batch of galls after soaking in water; the wheat grain nematode (Anguina tritici) came from the galls preserved in our laboratory in the 1970s, and the galls were shorter than healthy wheat grains And thick, nearly spherical, oily green at first, later turning yellowish brown to dark brown, with small hooks on the top, see attached picture 2; cut galls, and nematodes containing white filaments. Anguinawevelli was obtained from galls intercepted from teff seeds imported from South Africa on May 20, 2001. The galls are olive-shaped, dark brown, about twice the size of normal seeds, as shown in Figure 3, a large number of live larvae were isolated from this batch of galls after soaking in water; Ditylenchus destructor was reserved in our laboratory Live worm sample: Ditylenchus sp. in Langfang, Hebei Province. Ditylenchus isolated from diseased potatoes in Langfang, Hebei Province.

Experimental method: Use a picking needle to pick several living larvae one by one from the Nematode benthamum sample, wash them in deionized water for 3 times, and set aside. Add 2 μl deionized water to a clean glass slide, pick a washed nematode, cut the nematode into 3 sections with a scalpel, suck it into a 200 μl Eppendorf tube, and wash the scalpel and slide with 6 μl deionized water Finally, suck the washed water into the same Eppendorf tube; add 1.0 μl of 10-fold PCR buffer and 1.0 μl of proteinase K, and put it in an ultra-low temperature refrigerator for 20 minutes at -70°C. Take out the frozen nematodes, quickly put them into a PCR instrument or a water bath, and incubate at 65°C for 1 to 2 hours, then place the Eppendoff tube in a PCR instrument or a water bath, and incubate at 95°C for 10 minutes to lose proteinase K. active. Make 10 μl of DNA template.

A 50 μl PCR reaction system was used, which included: 8 μl DNA template, 5 μl 10-fold PCR buffer; 4 μl 25 mmol/L MgCl2; 2 μl 2.5 mmol/LdNTP; 1 μl primer sequence 1 at a concentration of 20 pmol/μl; 1 μl primer sequence 2 , the concentration is 20pmol/μl; 0.4μl Taq enzyme, 5U/μl; 28.6μl deionized water. Amplification was performed using a DNA Engine model PTC-200. The PCR reaction conditions are: (1) 94°C for 3 minutes; (2) 30 cycles in total, each cycle including three steps of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds; (3) 72°C for 5 seconds minute. The PCR reaction product was detected by electrophoresis to obtain a unique DNA band. The obtained PCR product was recovered and verified by sequencing, which proved that the obtained product was a specific product.

According to the gene sequence, primers, and taking into account specificity requirements, the TaqMan probe sequence was designed and synthesized as follows: 5'-TCATGTCTTGGCTATTGTAGACGTATCTGA-3', the 5' end of the probe is labeled with a FAM fluorescent reporter group, and the 3' end is connected to TAMRA to quench the fluorescence group.

At the same time, the obtained PCR product was recovered to construct a standard plasmid.

(2) preparing the DNA template of the sample to be tested;

Take the worms in the sample to be tested, pick out several live larvae from the sample one by one with a pick needle, put them into deionized water and wash them 3 times, and set them aside. Add 2 μl deionized water to a clean glass slide, pick a washed nematode, cut the nematode into 3 sections with a scalpel, suck it into a 200 μl Eppendorf tube, and wash the scalpel and slide with 6 μl deionized water Finally, suck the washing water into the same Eppendorf tube; add 1.0 μl of 10-fold PCR buffer and 1.0 μl of proteinase K, and put it in an ultra-low temperature refrigerator for 20 minutes at -70°C. Take out the frozen nematodes, quickly put them into a PCR instrument or a water bath, and incubate at 65°C for 1 to 2 hours, then place the Eppendorf tube in a PCR instrument or a water bath, and incubate at 95°C for 10 minutes to lose proteinase K. active. Make 10 μl of DNA template.

(3) Fluorescence PCR detection is carried out on the DNA template of the sample to be tested;

Perform fluorescent PCR detection on the sample DNA template, and the composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Concentration Sample Amount

DNA sample 1μL

PCR buffer 10 times 1μL

MgCl2 25mM 0.5μL

dNTP 10mM 0.2μL

Primer sequence 1 20μmol/L 0.2μL

Primer sequence 2 20μmol/L 0.2μL

TaqMan probe sequence 20μmol/L 0.1μL

Taq enzyme 5U/μl 0.1μL

Double distilled water 6.7μL

Total volume 10μL

The matching fluorescent PCR amplification program is

(1) 94°C for 3 minutes;

(2) 94°C for 10 seconds;

(3) 30 seconds at 60°C;

(4) Go back to procedure (2), repeat 45 times

Then carry out fluorescent PCR detection on the standard plasmid, and carry out positive control with the result of step (3) to obtain the detection result.

The present invention will be further described below in conjunction with specific examples.

Example 1

Take the worms in a sample to be tested, use a picking needle to pick several live larvae from the sample one by one, wash them in deionized water for 3 times, and set aside. Add 2 μl deionized water to a clean glass slide, pick a washed nematode, cut the nematode into 3 sections with a scalpel, suck it into a 200 μl Eppendorf tube, and wash the scalpel and slide with 6 μl deionized water Finally, suck water into the same Eppendorf tube; add 1.0 μl of 10-fold PCR buffer and 1.0 μl of proteinase K, and put it into an ultra-low temperature freezer for 20 minutes at -70°C. Take out the frozen nematodes, quickly put them into a PCR instrument or a water bath, and incubate at 65°C for 1 to 2 hours, then place the Eppendorf tube in a PCR instrument or a water bath, and incubate at 95°C for 10 minutes to lose proteinase K. active. Make 10 μl of DNA template.

Perform fluorescent PCR detection on the sample DNA template, and the composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Concentration Sample Amount

DNA sample 1μL

PCR buffer 10 times 1μL

MgCl2 25mM 0.5μL

dNTP 10mM 0.2μL

Primer sequence 1 20μmol/L 0.2μL

Primer sequence 2 20μmol/L 0.2μL

TaqMan probe sequence 20μmol/L 0.1μL

Taq enzyme 5U/μl 0.1μL

Double distilled water 6.7μL

The total volume is 10 μL.

The matching fluorescent PCR amplification program is:

(1) 94°C for 3 minutes

(2) 94°C for 10 seconds

(3) 30 seconds at 60°C

(4) Go back to step 2 and repeat 45 times.

Similarly, 1 μL of the reporter plasmid, that is, the standard plasmid, was also subjected to fluorescent PCR detection to prove the effectiveness of the detection. The composition ratio of each component in the PCR reaction system used to detect the plasmid is as follows:

Composition Concentration Concentration Sample Amount

Reporter plasmid 1 μL

PCR buffer 10 times 1μL

MgCl2 25mM 0.5μL

dNTP 10mM 0.2μL

Primer sequence 1 20μmol/L 0.2μL

Primer sequence 2 20μmol/L 0.2μL

TaqMan probe sequence 20μmol/L 0.1μL

Taq enzyme 5U/μl 0.1μL

Double distilled water 6.7μL

The total volume is 10 μL.

The accompanying fluorescent PCR amplification procedure is the same as the procedure for detecting the sample to be tested in this embodiment.

The test result is: the sample amplification result is automatically analyzed by the instrument, and an obvious amplification curve can be found, as shown in curve 1 in Figure 4. The abscissa in Figure 4 is cycle Number, and the ordinate is Fluorescence (F1). The test results showed that the sample contained Nematode bentgrass. The amplification results of the reporter plasmid were automatically analyzed by the instrument, and an obvious amplification curve could be found, as shown in curve 1p in Figure 4 . The test results showed that the positive reference for this test was valid.

At the same time, the conventional morphological method and PCR method were used for detection, and the results were obtained three days later to prove that the nematode in the sample was C. benthamiana.

Example 2

The worm body in a certain sample to be tested is taken, and the preparation method of the worm body DNA template is the same as in Example 1.

The DNA template of the sample is detected by fluorescent PCR, and the composition ratio of each component in the PCR reaction system and the fluorescent PCR amplification procedure are the same as those in Example 1. Similarly, 1 μL of the reporter plasmid was also tested by fluorescent PCR to prove the validity of the test. The constituent ratios of the components in the PCR reaction system used to detect the plasmid, the PCR amplification program and the sample detection program in this implementation are the same as in Example 1.

The detection result is that the sample amplification result is automatically analyzed by the instrument, and an obvious amplification curve can be found, as shown in curve 2 in Figure 4 . The test results showed that the sample contained Nematode bentgrass. The amplification results of the reporter plasmid were automatically analyzed by the instrument, and an obvious amplification curve could be found, as shown in curve 2p in Figure 4 . The test results showed that the positive reference for this test was valid.

At the same time, the conventional morphological method and PCR method were used for detection, and the results were obtained three days later to prove that the nematode in the sample was C. benthamiana.

Example 3

The worm body in a certain sample to be tested is taken, and the preparation method of the worm body DNA template is the same as in Example 1.

The DNA template of the sample is detected by fluorescent PCR, and the composition ratio of each component in the PCR reaction system and the fluorescent PCR amplification procedure are the same as those in Example 1. Similarly, 1 μL of the reporter plasmid was also tested by fluorescent PCR to prove the validity of the test. The constituent ratios of the components in the PCR reaction system used to detect the plasmid, the PCR amplification program and the sample detection program in this implementation are the same as in Example 1.

The test result is the sample amplification result, which is automatically analyzed by the instrument, and no amplification curve is found. The test results showed that the sample did not contain Nematode bentgrass. The amplification results of the reporter plasmid were automatically analyzed by the instrument, and an obvious amplification curve can be found, as shown in curve 1 in Figure 5, where the abscissa in Figure 5 is cycle Number, and the ordinate is Fluorescence (F1). The test results showed that the positive reference for this test was valid.

At the same time, conventional morphological methods and PCR methods were used for detection, and the results obtained after three days proved that the samples did not contain bentgrass nematodes, and the worms were Dityienchus destructor.

Example 4

The worm body in a certain sample to be tested is taken, and the preparation method of the worm body DNA template is the same as in Example 1.

The DNA template of the sample is detected by fluorescent PCR, and the composition ratio of each component in the PCR reaction system and the fluorescent PCR amplification procedure are the same as those in Example 1. Similarly, 1 μL of the reporter plasmid was also tested by fluorescent PCR to prove the validity of the test. The constituent ratios of the components in the PCR reaction system used to detect the plasmid, the PCR amplification program and the sample detection program in this implementation are the same as in Example 1.

The test result is the sample amplification result, which is automatically analyzed by the instrument, and no amplification curve is found. The test results showed that the sample did not contain Nematode bentgrass. The amplification results of the reporter plasmid were automatically analyzed by the instrument, and an obvious amplification curve could be found, as shown in curve 2 in Figure 5. The test results showed that the positive reference for this test was valid.

At the same time, the conventional morphological method and PCR method were used for detection, and the results obtained after three days proved that the sample did not contain Nematode bentgrass, and the insect body was Anguinatritici.

According to the above examples, it is proved that the real-time fluorescent PCR detection method for Nematode bentgrass described in the present invention has specificity, and can quickly and accurately detect Nematode bentgrass from samples.

SEQUENCE LISTING

<110>South China Agricultural University: Animal, Plant and Food Sub-Center of Tianjin Entry-Exit Inspection and Quarantine Bureau

<120>A method for identifying bentgrass nematodes using real-time fluorescent PCR technology

<130>

<160>1

<170>PatentIn version 3.2

<210>1

<211>89

<212>DNA

<213> Artificial sequence

<400>1

gtttgcctac cggttgttta cggccgtctt atcatgtctt ggctattgta gacgtatctg 60

atggctgttt tcacaccgac tgcatgtgg 89

Claims (4)

1. method of using real-time fluorescence PCR technical appraisement bent grass grain nematode is characterized in that may further comprise the steps:

(1) dna profiling of preparation standard product carries out pcr amplification, reclaims amplified production, order-checking, and the gene that obtains to have sequence shown in table SEQ ID NO:1 designs and synthesizes the TaqMan probe according to described gene and primer sequence, preparation standard product plasmid;

Described primer sequence is:

Primer sequence 1 '-CCACATGCAGTCGGTGTGAA-3;

Primer sequence 25 '-GTTTGCCTACCGGTTGTTTACG-3 ';

Described probe sequence is:

5’FAM-TCATGTCTTGGCTATTGTAGACGTATCTGA-TAMRA3’；

(2) dna profiling of preparation testing sample;

(3) the testing sample dna profiling being carried out fluorescent PCR detects;

(4) the standard items plasmid is carried out fluorescent PCR and detect, the result carries out positive control with step (3), obtains testing result.

2. according to the method for the described utilization real-time fluorescence PCR of claim 1 technical appraisement bent grass grain nematode, it is characterized in that the described pcr amplification condition of step (1) is: (1) 94 ℃, 3 minutes; (2) 30 circulations, wherein each circulation comprises 94 ℃ 30 seconds, 56 ℃ 30 seconds and 72 ℃ of 30 seconds three steps; (3) 72 ℃ 5 minutes.

3. according to the method for the described utilization real-time fluorescence PCR of claim 1 technical appraisement bent grass grain nematode, it is characterized in that each component composition is as follows in the PCR reaction system that the described PCR of step (3) or step (4) detects:

Constituent concentration application of sample amount

DNA sample 1 μ L;

10 times of 1 μ L of PCR buffer;

MgCl2 25mM 0.5μL；

dNTP 10mM 0.2μL；

Primer sequence one 20 μ mol/L 0.2 μ L;

Primer sequence 2 20 μ mol/L 0.2 μ L;

Probe sequence 20 μ mol/L 0.1 μ L;

Taq enzyme 5U/ μ l 0.1 μ L;

Distilled water 6.7 μ L;

Cumulative volume 10 μ L;

Wherein the DNA sample is the dna profiling of standard items plasmid or testing sample.

4. according to the method for the described utilization real-time fluorescence PCR of claim 1 technical appraisement bent grass grain nematode, it is characterized in that the described PCR of step (3) or step (4) detects amplification program and is:

(1) 94 ℃ 3 minutes;

(2) 94 ℃ 10 seconds;

(3) 60 ℃ 30 seconds;

(4) get back to program (2), repeat 45 times.

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