CN111100943A

CN111100943A - Single-tube nested PCR (polymerase chain reaction) primer pair for detecting sugarcane puccinia hancei and detection method

Info

Publication number: CN111100943A
Application number: CN202010045649.0A
Authority: CN
Inventors: 吴伟怀; 刘宝慧; 贺春萍; 易克贤; 梁艳琼; 习金根; 郑金龙; 李锐
Original assignee: CATAS Environment and Plant Protection Institute
Current assignee: CATAS Environment and Plant Protection Institute
Priority date: 2020-01-16
Filing date: 2020-01-16
Publication date: 2020-05-05
Anticipated expiration: 2040-01-16
Also published as: CN111100943B

Abstract

The invention discloses a single-tube nested primer pair and a method for detecting sugarcane puccinia thryngii. The specific primer pair consists of nucleotides shown as a sequence 1 in a sequence table, nucleotides shown as a sequence 2 in the sequence table, nucleotides shown as a sequence 3 in the sequence table and nucleotides shown as a sequence 4 in the sequence table. The detection method has higher sensitivity and meets the requirement of daily detection. Meanwhile, the detection primer pair provided by the invention can shorten the time for identifying the sugarcane ophiomorpha, and can identify the sugarcane ophiomorpha relatively quickly, so that the problems of complicated steps, long identification period and the like of the traditional identification method are solved.

Description

Single-tube nested PCR (polymerase chain reaction) primer pair for detecting sugarcane puccinia hancei and detection method

Technical Field

The invention relates to the field of epidemic monitoring of plant diseases, in particular to a single-tube nested PCR primer pair, a kit and a detection method for detecting sugarcane puccinia crustii (P.kuehniii).

Background

Yellow rust disease of sugarcane caused by D.cruellus (P.kuehnii) is a worldwide fungal disease [ Ryan CC, Egan B T.Rust.// Ricaud C, Egan B T, Gillaspie Jr AG, et al, eds. Diseases of Sugarcane: Major Diseases [ M ]. Amsterdam, the Netherlands: Elsevier,1989: 189-. The disease has been distributed primarily in Asia and Australia [ Ryan C, Egan B T.Rust.// Ricaud C, Egan BT, Gillaspie Jr AG, et al, eds. Diseases of Sugarane: Major Diseases [ M ]. Amsterdam, the Netherlands: Elsevier,1989:189-210 ], and recently has gradually spread to countries such as Florida, Crimala, Gossda Rica, Nigali, Salvado, Panama, Mexico, Brazil, Cuba, Columbia, Mumicaca, Guador, melon etc. and regions [ Amaresh Chandra, Amber T.Keierweerd, Michelel P.Grisha.Detion of Puccinia ] and Australia Biotechnology [ J-2016. (2016-58: Biotechnology J-2016, Biotechnology J.2016 ]. The yield caused by the yellow rust of the sugarcane is considered to be inferior to that of the brown rust of the sugarcane, and the yellow rust of the sugarcane is continuously diffused and is more than ever. At present, sugarcane rust diseases occur in Guangxi, Yunnan, Guangdong, Fujian, Sichuan, Hainan and the like in sugarcane planting areas in China, and complex infection phenomena [ Fuhua English, Shouchonghua, Liu Yinghang, and the like exist, molecular detection of sugarcane rust disease pathogenic bacteria and sugarcane strain brown rust resistance genes Bru1 [ J ]. the science of tropical crops, 2016, 37 (5): 958-963].

For the detection and identification of the rust fungus of the sugarcane, the conventional morphology is often relied on in the past and the traditional identification host method is combined. The method needs the processes of separation (obtaining) of pathogenic bacteria, morphological identification, tieback, re-separation and the like, wastes time and labor in the whole process, and needs professional taxonomy knowledge and rich experience. Nevertheless, the accuracy and reliability of the result are susceptible to external factors, and it is difficult to meet the actual requirements of rapid and high-throughput identification and detection. With the development of molecular biology techniques in recent years, molecular detection methods typified by PCR techniques have been rapidly developed and applied. The method has the characteristics of rapidness, accuracy and sensitivity and does not need to carry out separation culture of pathogenic bacteria. Currently, 2 conventional PCR detection methods specific to Ruscus saccharea [ [ Glynn NC, Dixon LJ, Castlebury LA, Castlebury LisaA, Comstock Jack. PCR assays for the research and study vaccine in P.kuehn [ J ]. Plant Pathology,2010,59(4):703 and 711 ], and the real-time fluorescent quantitative PCR molecular detection method of Ruscus saccharea (P.kuehn). Traditional PCR primers are designed aiming at the Erysiphe hancei (P.kuehnii) at the earlier stage of the laboratory, and a relatively reliable PCR detection method is established. However, the detection method still has the problems of low sensitivity, false positive and the like. Therefore, further development and optimization of the detection and monitoring technology of the sugarcane ophiomorpha is necessary.

The nested PCR detection technology is a molecular detection technology which is commonly used for detecting plant diseases due to higher sensitivity. However, since nested PCR requires 2 independent PCR amplification reactions, and the second PCR amplification reaction uses the first PCR amplification product as a template, the template transfer needs to be performed by opening the tube during the process, which increases the probability of contamination. Whereas single-tube nested PCR was developed on the basis of nested PCR. In contrast, the single tube nested PCR does not require the tube rotation step, thereby reducing the risk of contamination. This makes the single-tube nested PCR detection technique not only have the specificity and sensitivity of nested PCR detection technique, but also save time and cost, and reduce the risk of pollution, and has a better development prospect [ AloyceR C, Tairo F, Sseruwagi P, Rey M.E.C, Ndunguru J.Ashingle-tube double and dmultiplex PCR for single and multiple detection of fungal viral infection in nova plants [ J ] Journal of viral Methods,2013,189(1):148-156 ]. At present, the single-tube nested PCR detection technology has been applied to the detection of pathogenic fungi. However, this technique has not been reported on the sugar cane puccinia trindoniformis.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and detect the sugarcane puccinia cruenti (P.kuehniii) more quickly, accurately and practically. The invention provides a single-tube nested PCR primer pair of sugarcane puccinia cruzi (P.kuehniii) with good specificity and high sensitivity, and a molecular detection method with strong specificity, high sensitivity, easy operation and reliable result.

The invention firstly provides a group of single-tube nested PCR detection primer pairs for rapidly detecting the puccinia hancei (P.kuehniii), wherein the specific primer pair consists of a forward outer primer, a reverse outer primer, a forward inner primer and a reverse inner primer, wherein the forward outer primer is nucleotide shown as a sequence 1 in a sequence table, the reverse inner primer is nucleotide shown as a sequence 2 in the sequence table, the forward inner primer is nucleotide shown as a sequence 3 in the sequence table, and the reverse inner primer is nucleotide shown as a sequence 4 in the sequence table.

Specifically, the sequences of the primer pairs are shown as follows:

forward outer primer (PkF 1): 5'-AACTTGTTAATATGGGGGAAACCTCA-3' (sequence 1 in the sequence table);

reverse outer primer (PkR 1): 5'-TGTGTGTGTTTTTTTAGTAGTCCCATTC-3' (SEQ ID NO: 2 in the sequence Listing).

Forward inner primer (PkF 2): 5'-AAAATGGATGTTGAGTGTTGC-3' (SEQ ID NO: 3 in the sequence Listing);

reverse inner primer (PkR 2): 5'-CACCTTCCTTGATGTGATTTT-3' (SEQ ID NO: 4 in the sequence Listing).

The invention also aims to provide a single-tube nested PCR detection method for the sugarcane puccinia hancei (P.kuehniii), which has the advantages of good specificity, high sensitivity and simple operation.

The single-tube nested PCR detection method of the sugarcane ophiomorpha saccharina (P.kuehniii) of the invention takes sugarcane leaves to be detected or unknown pathogenic bacteria spore DNA as a template, applies the specific primer pair to carry out PCR amplification in a single PCR reaction tube, and detects a PCR product by agarose gel electrophoresis to obtain a target band of 136 bp.

The molar ratio of the forward outer primer to the forward inner primer is 1: 1; the molar ratio of the forward outer primer to the reverse inner primer is 1: 1; the final concentration ratio of the outer primer to the inner primer is 5 fM: 0.5. mu.M.

In the above PCR detection method, the PCR reaction system is: dd H₂O13.3. mu.L, 10rTaq Buffer 2. mu.L, 2.5mM/L d NTPs 1.6. mu.L, rTaq DNApolymerase 0.1. mu.L, 0.1pM PkF1/PkR 11. mu.L, 10. mu.M PkF2/PkR 21. mu.L, and template DNA to be tested 1. mu.L.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 10s, extension at 72 ℃ for 25s, 20 cycles, and extension at 72 ℃ for 4 min; pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 53 deg.C for 10s, extension at 72 deg.C for 25s, 45 cycles, extension at 72 deg.C for 4min, and storage at 12 deg.C.

The third aspect of the present invention provides a single-tube nested PCR detection method for rapidly detecting the strain of Puccinia hancei (P.kuehniii), which comprises the step of performing PCR amplification using the primer pair according to the first aspect of the present invention or the primer pair in the kit according to the second aspect of the present invention.

Further, the method comprises the steps of:

step 1, extracting DNA from a sample;

step 2, carrying out single-tube nested PCR amplification by using the primer pair of the first aspect or the primer pair in the kit of the second aspect to obtain an amplification product;

and 3, after the reaction is finished, detecting the amplified product by 1.2% gel electrophoresis, thereby judging whether the sample contains a 136bp specific band of the sugarcane puccinia hancei (P.kuehniii).

In the present invention, the method for extracting DNA from a sample to be tested is not particularly limited, and any known DNA extraction method or kit may be used.

In the present invention, the condition for performing PCR amplification on the template DNA of the sample to be tested is not particularly limited. According to some embodiments of the present invention, the PCR amplification reaction system (20 μ L): dd H₂O13.3. mu.L, 10rTaq Buffer 2. mu.L, 2.5mM/L d NTPs 1.6. mu.L, rTaq DNA Polymerase 0.1. mu.L, 0.1pM PkF1/R1 (mixture of equal volumes of nucleotides shown in

sequences

1 and 2 in the sequence table) 1. mu.L, and 10. mu.M PkF2/R2 (mixture of equal volumes of nucleotides shown in

sequences

3 and 4 in the sequence table) 1. mu.L.

Experiments prove that the single-tube nested kit for detecting the sugarcane yellow stalk rust (P.kuehnii) and the special primer pair thereof can specifically detect the sugarcane yellow stalk rust (P.kuehnii), and any bands can not be detected from DNA of other test strains, such as sugarcane brown rust (Puccinia melanotheca) and grape layer rust (Phakopsora ampelopsis). According to some specific examples of the invention, the invention provides a single-tube nested kit for detecting the sugarcane puccinia hancei (P.kuehniii) and a special primer thereof, wherein the minimum detection amount of the DNA of the sugarcane puccinia hancei (P.kuehniii) is 10 fg/. mu.L, which is 1,000 times higher than the sensitivity (10 pg/. mu.L) of the common PCR detection method. Has higher sensitivity and meets the requirement of daily detection. Meanwhile, the detection primer can shorten the time for identifying the sugarcane puccinia lundii (P.kuehniii), and can quickly identify the sugarcane puccinia lundii (P.kuehniii), thereby overcoming the problems of complicated steps, long identification period and the like of the traditional identification method. The primer pair disclosed by the invention has the characteristics of high sensitivity and strong specificity when used for detection, and the detection method disclosed by the invention has the advantages of high efficiency, rapidness and high sensitivity, and is particularly suitable for batch detection.

Drawings

FIG. 1 shows primers for single-tube nested PCR detection of P.cruehnii (P.kuehniii) of Ruscus alvarezii

FIG. 2 shows the results of the inner primer annealing temperature screening provided by the present invention

M, DL2000 standard molecular weight; CK, ddH₂O；1，52℃；2，53℃；3，54.7℃；4，57.3℃；5，60.9℃；6，63.2℃；7，64.9℃；8，66℃

FIG. 3 shows the results of the outer primer annealing temperature range screening provided by the present invention

M, DL2000 standard molecular weight; CK, ddH₂O；1，61℃；2，61.9℃；3，63.4℃；4，65.5℃；5，68.2℃；6，70.7℃；7，72.1℃；8，73℃

FIG. 4 shows the results of detecting the concentration of the outer and inner primers of the single-tube nested PCR

M, DL2000 standard molecular weight; CK, ddH₂O；1，10nM：10μM；2，10nM：5μM；3，10nM：1μM；4，1pM：10μM；5，1pM：5μM；6，1pM：1μM；7，0.1pM：10μM；8，0.1pM：5μM；9，0.1pM：1μM；10，1fM：10μM；11，1fM：5μM；12，1fM：1μM；13，0.01fM：10μM；14，0.01fM：5μM；15，0.01fM：1μM

FIG. 5 is a specific analysis of M, DL2000 standard molecular weight by single tube nested PCR detection technique of Puccinia hancei (P.kuehniii); CK, ddH₂O; 1-2, Puccinia cruentosa (Puccinia kuehniii); 3-4, Puccinia melanocarpus (Puccinia melanocarpala); 5, Puccinia albuminosa (Coleosporium plumeriae); phakopsora ampelopsis (Phakopsora ampelopsis); 7-8, rust bacteria of camelina coffea (hemieia vastatrix); 9-10, Colletotrichum species (Colletotrichum gloeosporioides); 11-12, brown spot fungus coffeacola (Cerosporacoffeicola); 13-14, Magnaporthe grisea; 15, ustilagososcitaminea (ustilagoscutaminea); 16, sisal stem rot (Aspergillus niger)

FIG. 6 shows the sensitivity results of general PCR detection of P.cruehnii (P.kuehniii).

M, DL2000 standard molecular weight; CK, ddH₂O；1，10ng/μL；2，1ng/μL；3，100pg/μL；4，10pg/μL；5，1pg/μL；6，100fg/μL；7，10fg/μL；8，1fg/μL

FIG. 7 shows the result of single-tube nested PCR sensitivity detection using primers provided by the present invention.

M, DL2000 standard molecular weight; CK, ddH₂O；1，10ng/μL；2，1ng/μL；3，100pg/μL；4，10pg/μL；5，1pg/μL；6,100fg/μL；7，10fg/μL；8，1fg/μL

Detailed Description

The invention will be better understood by reference to the following description, taken in conjunction with the accompanying drawings, which illustrate specific embodiments of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 preparation of a Single tube nested primer set for detection of Puccinia turmeroides (P.kuehniii)

1. Designing a primer:

the primer pair designed by the invention is specifically as follows:

an outer primer:

forward (PkF 1): 5'-AACTTGTTAATATGGGGGAAACCTCA-3' (sequence 1 in sequence table)

Reverse (PkR 1): 5'-TGTGTGTGTTTTTTTAGTAGTCCCATTC-3' (sequence 2 in sequence table)

An inner primer:

forward (PkF 2): 5'-AAAATGGATGTTGAGTGTTGC-3' (SEQ ID NO: 3 in the sequence Listing)

Reverse (PkR 2): 5'-CACCTTCCTTGATGTGATTTT-3' (SEQ ID NO: 4 in the sequence Listing)

The specific positions of the above primer design are shown in FIG. 1.

2. Primer synthesis

The designed primers PkF1, PkR1, PkF2 and PkR2 were assigned to the Guangzhou synthesis department of Enwei Weiji (Shanghai) trade Co., Ltd.

Example 2 Single tube nested PCR annealing temperature determination of Phosphaerella saccharina (P. kuehniii)

First, screening the optimal maximum fire temperature of the primer pair in the sugarcane ophiocordyceps (P.kuehniii)

Using the primer pair prepared in example 1, PCR gradient amplification was performed on a DNA template extracted from spores of Triperonospora sugarcanna (P.kuehniii).

Wherein, the reaction system of PCR amplification is 20 μ L, which is as follows:

the reaction procedure is as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 52-66 ℃ for 10s, extension at 72 ℃ for 25s, 35 cycles, extension at 72 ℃ for 4min, and storage at 12 ℃.

According to the experimental result, whether a 136bp band of the puccinia hancei (P.kuehniii) target is amplified or not is observed through gel imaging, so that the highest limit of the annealing temperature of the inner primer is judged. The results show that in 8 annealing temperatures tested, the inner primer PkF2/R2 can amplify specific target bands at the annealing temperature of 52 ℃ to 63.2 ℃, and the amplification bands gradually weaken along with the increase of the annealing temperature; no band was amplified at an annealing temperature of 64.9 ℃ (FIG. 2). Therefore, the maximum annealing temperature of the inner primer was 64.9 ℃. Accordingly, 53 ℃ was selected as the optimum annealing temperature for the inner primer.

Secondly, screening the optimal annealing temperature of the external primer pair of the sugarcane ophiocordyceps cruzi (P.kuehniii)

Using the primer pair prepared in example 1, a DNA template extracted from spores of Triperonospora sugarcanum (P.kuehnii) was subjected to gradient annealing temperature PCR amplification.

the reaction procedure is as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 61-73 ℃ for 10s, extension at 72 ℃ for 25s, 35 cycles, extension at 72 ℃ for 4min, and storage at 12 ℃.

According to the experimental result, whether a 517bp stripe of the puccinia hancei (P.kuehniii) is amplified or not is observed through gel imaging, and then the optimal annealing temperature of the outer primer is judged. The results showed that, among the 8 annealing temperatures tested, bands of the expected size were amplified at 61 ℃, 61.9 ℃, 63.4 ℃ and 65.5 ℃, while the remaining 4 annealing temperatures did not show any bands (FIG. 3). Based on the maximum annealing temperature of the inner primer of 64.9 ℃, and the basic rule of the annealing temperatures of the inner primer and the outer primer of the single-tube nested PCR, the optimal annealing temperature of the outer primer is determined to be 65 ℃.

Example 3: single-tube nested PCR external-internal primer concentration ratio screening of sugarcane puccinia hancei (P.kuehniii)

Based on the primer pair prepared in example 1 and the annealing temperatures of the inner and outer primers determined in example 2, the DNA template extracted from spores of Erysiphe sugarcanum (P.kuehniii) was screened for the optimum concentration ratio of the inner and outer primers.

The optimal concentration ratio of the C.canephora (P.kuehnii) was observed after 5 concentration gradients of 10nM, 1pM, 0.1pM, 1fM and 0.01fM for the inner primer concentration, 1. mu.M and 1. mu.M for the outer primer concentration, respectively, were cross-combined. A20. mu.L reaction was used: dd H₂O13.3. mu.L, 10R Taq Buffer 2. mu.L, 2.5mM/L d NTPs 1.6. mu.L, rTaq DNApolymerase 0.1. mu.L, PkF 1/R11. mu.L, PkF 2/R21. mu.L, template DNA 1. mu.L.

Experimental results the optimal ratio of the concentrations of the inner and outer primers was determined by observing whether the bands were the same size as expected and the bands were the clearest by gel imaging system. As shown in FIG. 4, the 15 sets of outer and inner primer concentration ratios tested all amplified the corresponding bands. And when the concentration ratio of the outer primer to the inner primer is 0.1 pM: at 10. mu.M, the band was most clear, so the optimal concentration of the outer primer was set to 0.1 pM. Thus, the final concentration ratio of the outer and inner primers was 5 fM: 0.5. mu.M.

Example 4: specific detection of single-tube nested PCR of P.cruehnii.C.for P.cruehnii.C.with the primer pair prepared in example 1, the internal and external primer annealing temperatures and their ratios optimized in examples 2 and 3 were used to detect P.cruchuii.C.; puccinia melanocarpus (Puccinia melanocarpala); coleosporium ovalicatum (Coleosporium plumeriae); phakopsora ampelopsis (Phakopsora ampelopsis); rust bacteria of camelina coffea (Hemileia vastatrix); rubber anthracnose (Colletotrichum gloeosporioides); brown spot fungus (Cerospora coffeicola); rice blast (Magnaporthe grisea); ustilagososcitaminea (ustilagoscutaminea); performing single-tube nested PCR amplification on DNA templates such as sisal stem rot (Aspergillus niger) and the like.

PCR reaction (20. mu.L): dd H₂O13.3. mu.L, 10r Taq Buffer 2. mu.L, 2.5mM/L d NTPs 1.6. mu.L, r Taq DNA Polymerase 0.1. mu.L, PkF1 solution with concentration of 0.1 pM/PkR 1 solution with concentration of 0.1pM 1. mu.L, PkF2 solution with concentration of 10. mu.M and PkR2 solution 1. mu.L, template DNA 1. mu.L.

The amplification procedure was: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 10s, extension at 72 ℃ for 25s, 20 cycles, and extension at 72 ℃ for 4 min; pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 53 deg.C for 10s, extension at 72 deg.C for 25s, 45 cycles, extension at 72 deg.C for 4min, and storage at 12 deg.C.

As a result of the experiment, the specificity was determined by visually observing whether a 136bp band was amplified. The results showed that the primer pair could detect only the desired band from the product whose template was DNA of Erythrophytrium sacchari, but no band was detected from the DNA of any other strain (FIG. 5), indicating that the primer pair could specifically detect Erythrophytrium sacchari (P.kuehnii).

Example 5: sensitivity results for general PCR detection of C.sacchari (P.kuehniii).

Sensitivity of C.canephora (P.kuehnii) was detected by ordinary PCR using the pair of inner primers PkF2/PkR2 prepared in example 1. Adjusting initial concentration of ITS plasmid DNA of the strain of the sugarcane ophiocordyceps hancei to 10 ng/. mu.L, and gradually diluting the initial concentration to 10 by the order of magnitude of 10^-6ng/. mu.L was used as a template for ordinary PCR amplification. The results showed that only a weak band was detected when the plasmid template concentration was 10 pg/. mu.L; when the concentration of the plasmid template was less than 10 pg/. mu.L, no band was detected (FIG. 6). The result shows that the ordinary PCR can specifically detect the genomic DNA concentration of the sugarcane puccinia hancei with the lowest limit of 10 pg/mu L and the lowest detection final concentration of 0.5 pg/mu L.

Example 6: sensitivity of Single tube nested PCR for detection of Puccinia hancei (P.kuehniii)

Using the primer pair prepared in example 1, the concentration of the E.canna DNA recombinant plasmid pEASY-ITS was adjusted to 10 ng/. mu.L, and the DNA was gradually diluted down to 10 by 10 orders of magnitude^-6ng/mu L is used as a template to carry out single-tube nested PCR amplification, and the sensitivity of the primer pair provided by the invention is analyzed. The PCR system and reaction procedure were the same as in example 4, and the PCR product was detected by electrophoresis on a 1.2% agarose gel. The result shows that when the concentration of the plasmid template is 10 fg/. mu.L, only a weak band is detected; when the concentration of the plasmid template was more than 10 fg/. mu.L, no band was detected (FIG. 7). Therefore, the primer pair disclosed by the invention can be used for specifically detecting the sugarcane puccinia thryngii, the minimum detection limit is 10 fg/mu L, and the minimum detection final concentration is 0.5 fg/mu L. Compared with example 5, the primer is 1,000 times higher, which shows that the single-tube nested primer pair of the invention has very high sensitivity.

Sequence listing

<110> institute for environmental and plant protection of tropical agricultural academy of sciences in China

<120> single-tube nested PCR primer pair for detecting sugarcane puccinia chrysosporium and detection method

<130>WHOI201001

<160>4

<170>Patent-In 3.5

<210>1

<211>26

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>1

aacttgttaa tatgggggaa acctca 26

<210>2

<211>21

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>2

aaaatggatg ttgagtgttg c 21

<210>3

<211>28

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

tgtgtgtgtt tttttagtag tcccattc 28

<210>4

<211>21

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>4

caccttcctt gatgtgattt t 21

Claims

1. A group of single-tube nested primer pairs for detecting the Puccinia cruehniii comprise nucleotides shown in a sequence 1 in a sequence table, nucleotides shown in a sequence 2 in the sequence table, nucleotides shown in a sequence 3 in the sequence table and nucleotides shown in a sequence 4 in the sequence table.

2. A kit for detecting hypocrea sugarcane (p.kuehniii), comprising the single-tube nested primer pair of claim 1.

3. The kit according to claim 2, characterized in that: the kit also comprises PCR reaction reagents.

4. Use of the single-tube nested primer pair of claim 1 and the kit of claim 2 or 3 for detecting puccinia hancei (p.kuehniii).

5. A method of detecting puccinia hancei (p.kuehniii) comprising the steps of:

1) extracting DNA of a sample to be detected;

2) using a sample DNA to be detected as a template, and carrying out PCR amplification by using the single-tube nested primer pair of claim 1;

3) the amplification product was detected by direct visual observation of gel electrophoresis to determine whether the sample contained D.canephora (P.kuehnii).

6. The method of claim 5, wherein:

the reaction system of the single-tube nested PCR amplification is 20 mu L, and comprises: dd H₂O13.3 muL, 10r Taq Buffer 2 muL, 2.5mM/L d NTPs 1.6 muL, rTaq DNA Polymerase 0.1 muL, a solution 1 muL containing the nucleotide represented by the sequence 1 in the sequence table with the concentration of 0.1pM and the nucleotide represented by the sequence 2 in the sequence table with the concentration of 0.1pM, a solution 1 muL containing the nucleotide represented by the sequence 3 in the sequence table with the concentration of 10 muM and the nucleotide represented by the sequence 4 in the sequence table with the concentration of 10 muM, and a sample DNA to be detected is 1 muL.

7. The method of claim 5, wherein: performing agarose electrophoresis detection on a single-tube nested amplification reaction product of a detected sample, observing the result under a gel imager, and if the size of the observed band is consistent with that of a target band, indicating that the detected sample contains the puccinia hancei (P.kuehniii); otherwise, the sample to be detected does not contain the sugarcane puccinia cruentosa (P.kuehnii).