CN109182574B

CN109182574B - Nested PCR kit and method for detecting albizia falcataria ulcer germs

Info

Publication number: CN109182574B
Application number: CN201811046238.2A
Authority: CN
Inventors: 纪春艳; 王伟; 徐振江; 刘洪�; 徐大高; 敖莉丝
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-09-07
Filing date: 2018-09-07
Publication date: 2021-04-23
Anticipated expiration: 2038-09-07
Also published as: CN109182574A

Abstract

The invention discloses a nested PCR kit and a method for detecting albizia falcataria ulcer germs. The kit comprises a group of nested PCR primer groups for detecting the acantholepis falcata, and the nested PCR primer groups consist of a first round amplification primer pair EF-688F, EF-986R and a second round specific amplification primer pair EF-AF and EF-AR; the nucleotide sequences of the EF-688F, EF-986R, EF-AF and the EF-AR are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 4. The kit has the advantages of strong specificity, high sensitivity and high accuracy, can accurately detect the canker in the albizia falcataria sample with no obvious symptoms or when the quantity of the canker is extremely small, has high detection speed, and is suitable for quickly detecting and monitoring the canker of albizia falcataria.

Description

Nested PCR kit and method for detecting albizia falcataria ulcer germs

Technical Field

The invention relates to the technical field of phytopathogen detection, in particular to a nested PCR kit and a method for detecting albizia falcataria ulcer germs.

Background

Albizia falcataria is a famous tropical fast-growing tree species in the world, and has the advantages of short operation cycle, rich wood fiber, strong toughness and easy additionThe artificial board is an excellent raw material for artificial boards, pulping and papermaking, and has wide application and high economic benefit and landscape value. The Guangdong has built the biggest jacaranda base for albizzia falcata in China, and with the increasing planting area of the jacaranda falcata artificial forest, in recent years, the jacaranda falcata has common diseases, and the jacaranda falcata is prepared from cacao color diplospora (a) cacao color diplosporaLasiodiplodia theobromae) The caused albizia falcataria canker is the most important disease of albizia falcataria planting areas, and is mainly harmful to young trees grown for 1-2 years, so that serious economic loss is caused. The cocoa trichlora is mainly distributed in tropical and subtropical regions, hosts are very wide, more than 500 plant diseases can be caused, and the diseases mainly enter through wounds to cause symptoms of ulcer, necrosis, fruit rot, root rot and the like. The symptoms of the cacao trichoderma asperellum infecting albizzia are mainly shown as follows: early symptoms are not obvious, the trees grow slowly, and some leaves are yellow; the epidermis is asymptomatic, the epidermis is scraped, and some phloem can be seen to brown; with the infection and expansion of pathogenic bacteria, the stems and branches of diseased trees present typical dark brown depressions, large spots of ulcers, poor growth, withered top and death in severe cases. The natural onset of albizia falcataria in the early stage of the canker is not easy to be found, and after the typical symptoms appear, the prevention and treatment measures are taken too late.

The albizia falcataria ulcer disease is systematically identified and reported for the first time by Chun Yan et al (2017) in recent years, and no relevant report is found about molecular detection of albizia falcataria diseases. At present, pathogenic bacteria separation, culture, identification, symptom identification and other methods are mainly adopted to detect the albizia falcataria ulcer germs, but the methods are time-consuming and difficult to accurately diagnose samples with early-stage unobtrusive diseases. The method is based on the establishment of effective prevention and control measures, so that an accurate, sensitive and rapid albizia falcataria ulcer germ molecule detection method is established, the occurrence and development of the ulcer disease are detected in time, and the method has important theoretical guidance and practical significance on the healthy production and disease prevention and control of albizia falcataria.

Disclosure of Invention

The invention provides a nested PCR kit and a method for detecting albizia falcataria ulcer germs, aiming at overcoming the defect that no molecular detection method for albizia falcataria ulcer germs exists in the prior art. The kit has the advantages of strong specificity, high sensitivity and high accuracy, can accurately detect the canker in the sample of the albizia falcataria with no obvious symptoms or when the quantity of the canker infected is extremely small, has high detection speed, and is suitable for quickly detecting and monitoring the canker of the albizia falcataria.

The invention aims to provide a nested PCR primer set for detecting albizia falcataria ulcer germs.

The invention also aims to provide application of the nested PCR primer group in preparation of a reagent or a kit for detecting albizia falcataria ulcer germs.

The invention also aims to provide a nested PCR kit for detecting albizia falcataria ulcer germs.

The invention also aims to provide a nested PCR detection method for albizia falcataria ulcer germs.

In order to achieve the purpose, the invention is realized by the following scheme:

a group of nested PCR primer groups for detecting the albizia falcataria ulcerosa, which consists of a first round of amplification primer pair EF-688F, EF-986R and a second round of specific amplification primer pair EF-AF and EF-AR; the nucleotide sequences of the EF-688F, EF-986R, EF-AF and the EF-AR are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 4.

EF-688F（SEQ ID NO:1）：5’-CGGTCACTTGATCTACAAGTGC-3’

EF-986R（SEQ ID NO:2）：5’-TACTTGAAGGAACCCTTACC-3’

EF-AF（SEQ ID NO:3）：5’-GAGAAGTTCGAGAAGGTCCGTGCAC-3’

EF-AR（SEQ ID NO:4）：5’-CGTTAGCCATTGCTCGTACGACGAT-3’

The nested PCR is to use two pairs of PCR primers and two PCR cycles to amplify a target DNA fragment, has the advantages of high sensitivity, specificity and accuracy, and at present, no method for detecting albizia falcataria ulcerosa-cacao trichoderma by using the nested PCR exists at home and abroad. The sequence of the elongation factor-1 alpha protein gene (EF-1 alpha) has specificity among families, genera and different species in the genera, and has conservation in the species.

Therefore, the invention requests to protect the application of the nested PCR primer group in the preparation of a reagent or a kit for detecting the albizia falcataria ulcer germs.

The invention also claims a nested PCR kit for detecting the albizia falcataria ulcer germs, which comprises the nested PCR primer set for detecting the albizia falcataria ulcer germs.

Preferably, the final concentration ratio of the first round amplification primer pair and the second round specific amplification primer pair is 1: 1.

Preferably, the nested PCR kit further comprises a first round PCR reaction system; the first round PCR reaction system comprises 2 mu L of DNA template, 5 mu L of 10 XPCR Buffer, 4 mu L of dNTPs mixed solution, 0.25 mu L of 5U/mu L rTaq polymerase, 1 mu L of 10 mu moL L/L first round amplification primer pair and the balance of sterilized ultrapure water which is complemented to 50 mu L.

Preferably, the conditions of the first round of PCR reaction are pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 54 ℃ for 30 s, extension at 72 ℃ for 2 min, 25 cycles in total, and extension at 72 ℃ for 10 min; storing at 4 ℃.

Preferably, the nested PCR kit further comprises a second round PCR reaction system; the second round PCR reaction system comprises 2 mu L of products obtained by the first round PCR reaction, 5 mu L of 10 XPCR Buffer, 4 mu L of dNTPs mixed solution, 0.25 mu L of 5U/mu L rTaq polymerase, 1 mu L of second round specific amplification primer pair of 10 mu moL L/L and the balance of sterilized ultrapure water which is complemented to 50 mu L.

Preferably, the conditions of the second round of PCR reaction are pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 63 ℃ for 30 s, and extension at 72 ℃ for 18 s for 37 cycles and extension at 72 ℃ for 7 min.

The invention also provides a nested PCR detection method for albizia falcataria ulcer germs, which comprises the following steps:

s1, extracting DNA of a tissue sample to be detected;

s2, performing nested PCR reaction by using the nested PCR kit;

and S3, carrying out electrophoresis on the reaction product, and if the electrophoresis result has a single target strip of 270 bp, judging that the tissue sample to be detected contains albizia falcataria ulcer germs.

Compared with the prior art, the invention has the following beneficial effects:

(1) the kit disclosed by the invention is high in detection sensitivity, the minimum detection limit can reach 100 ag/muL of genome DNA of albizia falcataria, and samples with no obvious disease sample or little quantity of infected ulcer disease bacteria can be accurately detected.

(2) The kit can amplify a single target strip with the size of 270 bp aiming at albizia falcataria ulcer germ genome DNA, and has strong specificity; other fungus strains have no amplification band, are not interfered by other pathogenic fungi, and have high accuracy.

(3) The detection method has the advantages of simple operation, short time consumption, high detection speed and strong practicability, and can judge the result after DNA extraction, PCR amplification and agarose electrophoresis and complete the detection within 8 hours. The kit is suitable for detecting the albizia falcataria ulcer germs and diagnosing the albizia falcataria ulcer, and is particularly suitable for early diagnosis.

Drawings

FIG. 1 is an electrophoretogram at different annealing temperatures in example 1; wherein, Lane M is DNA Marker; lane 1 is 55 ℃; lane 2 at 59 ℃; lane 3 is 63 ℃; lane 4 at 67 ℃; lane 5 at 71 ℃; lane 6 is water.

FIG. 2 is an electrophoretogram at different numbers of cycles in example 1; wherein, Lane M is DNA Marker; lane 1 is 22 cycles; lane 2 is 27 cycles; lane 3 is 32 cycles; lane 4 is 37 cycles; lane 5 is 42 cycles; lane 6 is water.

FIG. 3 is a specific electrophoretogram of nested PCR in example 2; wherein, Lane M is DNA Marker; lane 1 isL. theobromaeStrain BL 1331; lane 2 isL. theobromaeStrain HD 1332; lane 3 isRugonectria rugulosa(ii) a Lane 4 isPestalotiopsissp.(ii) a Lane 5 isLasiodiplodia pseudotheobromae(ii) a Lane 6 isNeofussicoccum parvum(ii) a Lane 7 isFusicoccum aesculi(ii) a Lane 8 isColletotrichum gloeosporioides(ii) a Lane 9 isXylariasp.(ii) a Lane 10 isPeronophythora litchii(ii) a Lane 11 is water.

FIG. 4 is a sensitive electrophoretogram of nested PCR in example 2; wherein, Lane M is DNA Marker; the mass concentrations of DNA in lanes 1-9 were 1 ng/. mu.L, 100 pg/. mu.L, 10 pg/. mu.L, 1 pg/. mu.L, 100 fg/. mu.L, 10 fg/. mu.L, 1 fg/. mu.L, 100 ag/. mu.L, and 10 ag/. mu.L, respectively; lane 10 is water.

FIG. 5 is the electrophoretogram of forest disease detection by nested PCR in example 3; wherein, Lane M is DNA Marker; lanes 1-5 are woodland jacaranda ulcerosis specimens; lane 6 isL. theobromaeStrain BL 1331; lane 7 isL. theobromaeStrain HD 1332; lane 8 is albizzia falcata healthy sample; lane 9 is water.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Embodiment 1 nested PCR kit and method for detecting Albizia falcata Miq ulcer germs

A nested PCR kit for detecting albizia falcataria ulcer germs comprises a nested PCR primer group for detecting albizia falcataria ulcer germs, a first round PCR reaction system and a second round PCR reaction system.

The nested PCR primer group for detecting the falcarica falcata canker is composed of a first round of amplification primer pair EF-688F, EF-986R and a second round of specific amplification primer pair EF-AF and EF-AR; the nucleotide sequences of the EF-688F, EF-986R, EF-AF and the EF-AR are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 4. The volume of the first round of amplification primer pair (10 mu moL/L) in the kit is 1 mu L, and the volume of the second round of specific amplification primer pair (10 mu moL/L) in the kit is 1 mu L.

EF-688F（SEQ ID NO:1）：5’-CGGTCACTTGATCTACAAGTGC-3’

EF-986R（SEQ ID NO:2）：5’-TACTTGAAGGAACCCTTACC-3’

EF-AF（SEQ ID NO:3）：5’-GAGAAGTTCGAGAAGGTCCGTGCAC-3’

EF-AR（SEQ ID NO:4）：5’-CGTTAGCCATTGCTCGTACGACGAT-3’

The first round of PCR reaction system comprises 2 mu L of DNA template, 5 mu L of 10 XPCR Buffer, 4 mu L of dNTPs mixed solution, 0.25 mu L of 5U/mu L rTaq polymerase, 0.5 mu L of each primer EF-688F/EF-986R (10 mu moL/L), and the balance of sterilized ultrapure water which is supplemented to 50 mu L.

The conditions of the first round of PCR reaction are pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 54 ℃ for 30 s, extension at 72 ℃ for 2 min, 25 cycles in total, and extension at 72 ℃ for 10 min; storing at 4 ℃.

The second round of PCR reaction system comprises 2 mu L of product obtained by the first round of PCR reaction, 5 mu L of 10 XPCR Buffer, 4 mu L of dNTPs mixed solution, 0.25 mu L of 5U/mu L of rTaq polymerase, 0.5 mu L of each primer EF-AF/EF-AR (10 mu moL/L), and the balance of sterilized ultrapure water which is complemented to 50 mu L.

The conditions of the second round of PCR reaction are pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 63 ℃ for 30 s, and extension at 72 ℃ for 18 s, wherein the conditions comprise 37 cycles and extension at 72 ℃ for 7 min.

The conditions of the second round of PCR reaction are optimized in the embodiment, and the conditions mainly comprise the following two aspects:

1. optimization for nest detection of annealing temperature of second round PCR reaction

And (3) selecting genome DNA of the albizia falcataria bacterial strain BL1331 as a template, and optimizing annealing temperature for the second round reaction condition of nested PCR detection. The reaction temperature was set in the following order: gradient experiments were performed at 55 deg.C, 59 deg.C, 63 deg.C, 67 deg.C and 71 deg.C, with a number of reaction cycles of 35 cycles to determine the optimum annealing temperature.

The annealing temperature optimization test results show (as shown in fig. 1): under the conditions that the annealing temperature of the nested second round PCR reaction is 55 ℃, 59 ℃, 63 ℃, 67 ℃ and 71 ℃, single amplification bands can be obtained, but the amplification band is brightest under the condition that the annealing temperature is 63 ℃, and the amplification band is dull under the condition that the annealing temperature is 71 ℃. Therefore, in order to obtain stable amplification, the optimal annealing temperature for the nested detection second round of PCR is 63 ℃.

2. Optimization of nest type detection second round PCR reaction cycle number

The number of reaction cycles is directly related to the number of amplification products, the genome DNA of the albizia falcataria bacterial strain BL1331 is selected as a template, and cycle number optimization is carried out on the second round reaction conditions of nested PCR. The number of reaction cycles was set as follows: 22. 27, 32, 37 and 42 were subjected to gradient tests at an annealing temperature of 63 c to determine the optimum number of cycles.

Cycle number optimization test results show (as shown in fig. 2): under the condition of the cycle number of 22-42, a single target amplification band is generated, and under the condition of the cycle number of 37, the amplification band is brightest, so that the optimal cycle number of nested detection second round PCR is determined to be 37 cycles.

A nested PCR detection method for albizia falcataria ulcer germs comprises the following steps:

s1, extracting DNA of a tissue sample to be detected;

s2, performing nested PCR reaction by using the nested PCR kit;

Example 2 kit Performance test

The nested PCR kit for detecting the albizia falcataria ulcer pathogen described in example 1 was tested for specificity and sensitivity.

1. Kit specificity detection

Reference fungal strains (common fungi on albizia falcataria and common pathogenic fungi on forest trees): crinkled Shell (Rugonectria rugulosa) Pestalotiopsis (A) PestalotiopsisPestalotiopsissp.), Erythrococos pseudotheobroma (C.pseudotheobroma: (C.pseudotheobroma) ((C.))Lasiodiplodia pseudotheobromae) Fusarium oxysporum (F. sp.) (C. sp.) (Neofussicoccum parvum) Fusarium septorium (F. aestivum)Fusicoccum aesculi) Colletotrichum gloeosporioides (B)Colletotrichum gloeosporioides) Xylaria (A. charri) (A. charred)Xylariasp.), Phytophthora litchi, (Phytophthora litchi), (Phytophthora litchiPeronophythora litchii）。

The method comprises the following specific steps:

(1) hyphal DNA extraction of test strains

Inoculating the tested fungus strain into a liquid PDA culture solution, carrying out shake cultivation for 3-5 d at 28 ℃ and 120 rpm, and filtering and collecting mycelia. Fungal hyphal DNA was extracted using a Fungal DNA Kit (OMEGA) and the DNA obtained was stored at-20 ℃ for later use.

(2) First round PCR amplification

And (2) performing first round PCR amplification by using the fungal hypha DNA obtained in the step (1) as a template and adopting a translation elongation factor-1 alpha protein (TEF-1 alpha) primer EF-688F/EF-986R.

First round PCR reaction System: mu.L of DNA template, 5. mu.L of 10 XPCR Buffer, 4. mu.L of dNTPs mixture, 0.25. mu.L of rTaq polymerase (5U/. mu.L), 0.5. mu.L of each primer EF-688F/EF-986R (10. mu.mol/L), and sterilized ultrapure water to make up to 50. mu.L.

First round PCR reaction procedure: pre-denaturation at 94 deg.C for 5 min, denaturation at 94 deg.C for 30 s, annealing at 54 deg.C for 30 s, and extension at 72 deg.C for 2 min for 25 cycles, and extension at 72 deg.C for 10 min; storing at 4 ℃.

(3) Second round of PCR amplification

And (3) taking the amplification product of the first round of PCR in the step (2) as a DNA template for the second round of PCR amplification, and performing the second round of PCR amplification by adopting a primer EF-AF/EF-AR.

Second round PCR reaction System: mu.L of DNA template, 5. mu.L of 10 XPCR Buffer, 4. mu.L of dNTPs mixture, 0.25. mu.L of rTaq polymerase (5U/. mu.L), 0.5. mu.L of each primer EF-AF/EF-AR (10. mu.mol/L), and a sterilized ultrapure water content of 50. mu.L.

Second round PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 63 ℃ for 30 s, and extension at 72 ℃ for 18 s for 37 cycles, and extension at 72 ℃ for 7 min.

(4) PCR amplification product detection

And (4) detecting the second round PCR amplification product obtained in the step (3), carrying out electrophoresis on the PCR product in 2% agarose gel by taking 5 mu L, carrying out electrophoresis in the electrophoresis buffer solution of 1 × TAE and under the voltage of 5V/cm, and observing the product under a gel imaging system or an ultraviolet lamp.

The results of the determination of the presence or absence of the target monospecific fragment (270 bp) in FIG. 3 show that: the albizia falcataria canker strains BL1331 and HD1332 (lanes 1-2) are positive, while other reference fungus strain samples (lanes 3-10) have no target fragment (270 bp), and the negative control water does not detect the target fragment. The established nested PCR can be determined to be capable of accurately and specifically detecting the jacaranda albiziae canker germ, namely, the cacao trichoderma.

2. Kit sensitivity detection

Test strains: strain BL1331 of trichoderma theobromae.

The method comprises the following specific steps:

(1) hyphal DNA extraction of the Strain

Inoculating the strain BL133 of the cacao trichoderma in the liquid PDA culture solution, performing shake culture at 28 ℃ and 120 rpm for 5 d, and filtering to collect mycelia. Fungal hyphal DNA was extracted using a Fungal DNA Kit (OMEGA) and the DNA obtained was stored at-20 ℃ for later use.

(2) First round PCR amplification

Mycelium DNA of the strain BL1331 is used as a template, and the mycelium DNA template is sequentially diluted by 10 times after the concentration is measured, so that the mass concentration gradients are sequentially 1 ng/mu L, 100 pg/mu L, 10 pg/mu L, 1 pg/mu L, 100 fg/mu L, 10 fg/mu L, 1 fg/mu L, 100 ag/mu L and 10 ag/mu L. With sterilized ddH₂O is a negative control. mu.L of each was used as a template, and first PCR amplification was performed using the translation elongation factor-1. alpha. protein (TEF-1. alpha.) primer EF-688F/EF-986R.

(3) Second round of PCR amplification

(4) PCR amplification product detection

According to the results of the detection of the presence or absence of the target monospecific fragment (270 bp) in FIG. 4, it was found that 1 specific band of 270 bp can be stably amplified using DNA having a mass concentration of 1 ng/. mu.L to 100 ag/. mu.L as a template, and no band is generated when DNA having a mass concentration of 10 ag/. mu.L is used as a template. The nested PCR reaction system can detect the cocoa mass concentration of 100 ag/mu L of the genome DNA of the trichoderma harynovii at the lowest, has high detection sensitivity and can meet the detection requirement of forest disease samples.

Example 3 nested PCR detection of woodland south Farneria albiziae ulcer disease samples

The kit of embodiment 1 is used for detecting the forest jacaranda mimosa canker disease sample, and the specific steps are as follows:

1. extraction of forest albizia falcataria ulcer disease-like tissue DNA

5 parts of suspected albizia falcataria ulcer disease sample and a healthy albizia falcataria plant sample collected from bos and huidong, wherein the 5 parts of suspected albizia falcataria ulcer disease sample comprise: disease sample 1: collected from Porro, with typical dark brown pits, ulcerated lesions; disease sample 2 and disease sample 3: from Palo, trees grow slowly, the epidermis is not obvious, and the phloem turns light brown; disease sample 4: collected from Huidong, with typical dark brown pits, ulcerated lesions; disease sample 5: collected from Huidong, trees grow slowly, leaves are yellow, and epidermis is not obvious; the phloem turns brown. About 100 mg of tissue was cut with a razor blade, the surface was sterilized with 75% ethanol for 30 seconds, washed three times with sterile water, blotted dry with sterile filter paper, placed in a mortar, added with liquid nitrogen, and rapidly pulverized into powder, and DNA extraction was performed according to the instructions of the Plant DNA Kit (OMEGA Co.). The DNA obtained can be stored at-20 ℃ for further use.

2. First round PCR amplification

Taking plant tissue sample DNA and albizia falcataria L.var.falcataria strain mycelium DNA as templates, and sterilizing ddH₂O is a negative control. mu.L of each was used as a template, and first PCR amplification was performed using the translation elongation factor-1. alpha. protein (TEF-1. alpha.) primer EF-688F/EF-986R.

3. Second round of PCR amplification

And (3) taking the amplification product of the first round of PCR in the step 2 as a DNA template for the second round of PCR amplification, and performing the second round of PCR amplification by adopting a primer EF-AF/EF-AR.

4. PCR amplification product detection

And (3) detecting the second round PCR amplification product obtained in the step (3), carrying out electrophoresis on 5 mu L of the PCR product in 2% agarose gel, carrying out electrophoresis in a 1 xTAE electrophoresis buffer solution at a voltage of 5V/cm, and observing the product under a gel imaging system or an ultraviolet lamp.

Nested PCR detection was performed using forest disease-like genomic DNA as a template according to the determination of the presence or absence of a single specific target fragment (270 bp) in FIG. 5. The result shows that all suspected albizia falcataria ulcer disease samples can be amplified to form a band of about 270 bp, and the band is bright. The positive control (albizia falcataria ulcer germ strains BL1331 and HD 1332) can amplify a single bright strip (270 bp), and the negative control (albizia falcataria healthy tissue sample and water) has no strip amplification, which shows that the nested PCR system can accurately and effectively detect the albizia falcataria ulcer germ in the forest.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

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Claims

1. A group of nested PCR primer groups for detecting the acanthopanax falcata canker is characterized by consisting of a first round of amplification primer pair EF-688F, EF-986R and a second round of specific amplification primer pair EF-AF and EF-AR; the nucleotide sequences of the EF-688F, EF-986R, EF-AF and the EF-AR are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 4.

2. Use of the nested PCR primer set of claim 1 in the preparation of a reagent or kit for detecting albizia falcataria l.f.

3. A nested PCR kit for detecting albizia falcataria ulcer germs of claim 1, comprising the nested PCR primer set for detecting albizia falcataria ulcers germs of claim 1.

4. A nested PCR kit according to claim 3, wherein the final concentration ratio of the first round amplification primer pair and the second round specific amplification primer pair is 1: 1.

5. A nested PCR kit according to claim 3, wherein the nested PCR kit further comprises a first round PCR reaction system; the first round PCR reaction system comprises 2 mu L of DNA template, 5 mu L of 10 XPCR Buffer, 4 mu L of dNTPs mixed solution, 0.25 mu L of 5U/mu L rTaq polymerase, 1 mu L of 10 mu moL L/L first round amplification primer pair and the balance of sterilized ultrapure water which is complemented to 50 mu L.

6. A nested PCR kit as claimed in claim 5 wherein the conditions of the first round of PCR reaction are pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 54 ℃ for 30 s, extension at 72 ℃ for 2 min for 25 cycles, and extension at 72 ℃ for 10 min; storing at 4 ℃.

7. A nested PCR kit according to claim 5, wherein the nested PCR kit further comprises a second round PCR reaction system; the second round PCR reaction system comprises 2 mu L of products obtained by the first round PCR reaction, 5 mu L of 10 XPCR Buffer, 4 mu L of dNTPs mixed solution, 0.25 mu L of 5U/mu L rTaq polymerase, 1 mu L of second round specific amplification primer pair of 10 mu moL L/L and the balance of sterilized ultrapure water which is complemented to 50 mu L.

8. A nested PCR kit as claimed in claim 7 wherein the conditions for the second round of PCR reaction are 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 30 s, 63 ℃ annealing for 30 s, 72 ℃ extension for 18 s for 37 cycles, and 72 ℃ extension for 7 min.

9. A nested PCR detection method for albizia falcataria ulcer germs is characterized by comprising the following steps:

s1, extracting DNA of a tissue sample to be detected;

s2, carrying out nested PCR reaction by using the nested PCR kit according to any one of claims 3-8;