CN1763193A - rDNA-ITS sequence of D. destructor in potato, specific detection primers and one-step double PCR detection method - Google Patents

Abstract

The present invention relates to " potato rot nematode rDNA-ITS sequence, specific detection primer and an one-step dual PCR detection method ".Potato rot nematode rDNA-ITS, it has the nucleotide sequence shown in SEQ NO1 or SEQ NO2; Specific detection primer SSsj at this rDNA-ITS sequences Design combines with universal primer AB28, add interior label primer D3A and D3B simultaneously, utilize an one-step dual PCR method to detect, the pcr amplification result of potato haulm nematode can obtain specificity 623bp fragment and 345bp fragment, and the pcr amplification result of other Ditylenchus dipsaci can only obtain the 345bp fragment.This invention provides molecular detecting method fast and accurately to the identification for Ditylenchus dipsaci in the quarantine and examination of Ditylenchus dipsaci and the production practice, provides theoretical reference for formulating Ditylenchus dipsaci disease comprehensive regulation decision-making.

Description

rDNA-ITS sequence of D. destructor potato, specific detection primers and one-step double PCR detection method

Technical field:

The invention belongs to the field of biological technology, and relates to the rDNA-ITS sequence of D. destructor potato, a specific primer sequence designed for the sequence and a rapid PCR detection method for D. destructor potato by using the primer.

technical background

D.potato rot nematode (D.destructor) which damages sweet potato and D. dipsaci which damages garlic, corms and bulbs are important diseases of crops in my country. Sweet potato stem nematode disease has been reported in 21 counties and cities including Lulong County, Changli, Luan County, Qian'an, Langfang, Bazhou, Quyang County and Dingzhou in Hebei, Miyun, Daxing, Fangshan in Beijing, Xuchang in Henan, Zhengzhou, Pingdingshan, Jia County, Jilin City, Jilin City, Liaoning, Shandong Yishui County, Jiangsu Xuzhou and other provinces and cities are common, and the disease is very serious, mainly infecting the underground roots, tubers and tubers of sweet potatoes, and the damaged parts show tissue necrosis, dryness Shrinkage, browning and cracking of bran core and epidermis, commonly known as sweet potato bran rot nematode disease, the disease generally reduces production by 20% to 30%, and in severe cases 40 to 50%, or even extinction, is a devastating nematode of sweet potato in North China and East China Disease, seriously restricted the development of sweet potato industry (Chen Pinsan, 1979, sweet potato stem nematode disease. Chinese crop diseases and insect pests (volume one). P460-465. Agricultural Press; Ye Songzhi Li Yuejie Liu Haiyuan The occurrence and control technology of sweet potato stem nematode disease Plant protection technology and Extension 199717(3), 20-21; Chen Fawei, Han Xianghong, and Li Xiangsheng. 1996. Integrated Control Technology of Sweet Potato Stem Nematode Disease Plant Protection Technology and Extension, 16(6), 12. Liu Xinyi, Hebei Province, Using Aldicarb to Control Peanut and Sweet Potato Nematodes Current status of the disease Plant Protection Technology and Extension 199717(3), 46~47. Lin Maosong Wenling Fang Zhongda Potato rot nematode and sweet potato stem nematode disease Jiangsu Agricultural Science Journal 199915(3), 186~190.).

The pathogen of sweet potato stem nematode disease in China, before the 1980s, was once identified as Ditylenchus dipsaci (Ditylenchus dipsaci) (Chen Pinsan, 1979, Sweet potato stem nematode disease. Chinese crop pests and diseases (volume 1). P460-465. Agricultural Press) Yin Guangde and Zhang Yunmei (1983) studied two standard samples of sweet potato stem nematode disease in Hebei and Shandong, and re-corrected the causative agent of sweet potato stem nematode disease as Ditylenchus destructor (Yin Guangde Zhang Yunmei 1983 The origin nematode of sweet potato stem nematode disease Revised. Shandong University Journal (Natural Science Edition), 1983, 9 (3): 117-127.), but the etiology of sweet potato stem nematode disease in other areas of China has not been studied in detail, so far the pathogen of sweet potato stem nematode disease in China is consistent and general Followed by D. destructor of potato.

With the adjustment of my country's agricultural industrial structure and the increase of multiple cropping index, the occurrence and harm of sweet potato stem nematode disease has increased year by year, and the disease of other economic crops planted after the new restructuring has become increasingly prominent, such as my country's Gansu, Ningxia, Hebei, Henan, Beijing, Inner Mongolia The occurrence of stem nematode diseases in other economic crops such as angelica, ginseng, astragalus, ephedra, mint, flax, etc. is becoming more and more serious, and it is urgent to diagnose quickly and formulate correct control countermeasures. Therefore, how to accurately, timely and quickly identify and identify mixed populations of D. nematodes in the field to the level of nematode species and subspecies has become an urgent primary problem for making comprehensive management decisions for plant D. nematode diseases.

In the past, the identification of stem nematode species was mainly based on morphological identification, and the identification of physiological races was mainly based on the pathogenicity of different hosts. Morphological identification of D. nematode is very difficult, there are not many effective identification features, and many phenotypic features are unstable. PCR-based molecular biology technology provides the most powerful tool for solving the problems of identification and diagnosis of stem nematode. Direct determination of genetic material (DNA) of nematode has become the most powerful tool for molecular diagnosis of nematode. In the molecular diagnosis and identification of plant nematodes, one of the most valuable genomic regions is the ribosomal DNA (ribosomal DNA, rDNA) repeat unit, rDNA has multiple copies (100-500) in the genome, and the internal transcription spacer (Internal Transcribed Spacer Region, ITS) sequence is very useful for nematode species and subspecies level molecular diagnosis, and is highly valued and widely used by nematologists. At present, the rDNA-ITS analysis technology has been widely used in Xiphinema, Grostochiensis, G.pallida, Heterodera, Aphelenchoides and Stem nematodes ( Ditylenchus), root-knot nematodes (Meloidogyne), root-rot nematodes (Pratylenchus), stinging nematodes (Belonolaimus), entomopathogenic nematodes, and aphids have been widely used in the study of molecular characteristics and molecular diagnostics of species and interspecies.

Ibrahim, S.K. et al. (1994) used two primers 18S and 26S to apply PCR technology to study the rDNA-ITS of 12 species of S. elegans and Stem nematode sativa, and amplified the ITS fragments of 12 species of S. elegans with a size of 860-1100bp , revealing the intraspecific and interspecific variation of S. rice, the ITS fragment amplified from the rice stem nematode is 1100bp (Ibrahim, S.K., Perry, R.N., Burrows, P.R.& Hooper, D.J.1994.Differentiation of species and populations of Aphelenchoides and of Ditylenchus angustus using a fragment of ribosomal DNA. Journal of Nematology 26, 412-421.). Wendt, K.R. et al. (1993) amplified a 900bp DNA fragment from D. sativa, D. phage and D. tweedes with universal primers rDNA1 and rDNA2, amplified from D. destructor (D.destructor) The resulting ITS fragment is 1200bp (Wendt, K.R, Vrain, T.C. & Webster, J.M.1993.Separation of three species of Ditylenchus and some host races of D.dipsaci by restriction fragment length polymorphism. Journal of Nematology 25:555-56).

Specific primer PCR or multiplex PCR technology is an important progress in nematode molecular diagnosis in recent years. One or more nematode species can be specifically detected in a mixed sample with a single PCR test. Nematode-specific primers based on ribosomal DNA (rDNA) have progressed rapidly, and have made breakthroughs in the diagnosis of root rot nematodes, root-knot nematodes, potato golden nematodes and potato white nematodes, soybean cyst nematodes and sugar beet cyst nematodes Progress, formed the specific diagnosis of 4 important root rot nematodes such as P. penetrans, P. scribneri, P. Coffeae and P. loosi. Sexual primers, and three kinds of root-knot nematodes such as M.Chitwoodi, M.fallax and M.Hapla (Zijlssstra C.2000, Identification of Meloidogyne chitwoodi, M. fallax and M.hapla based on SCAR-PCR: a powerful way of enabling reliable identification of populations or individuals that share common traits〔J〕European Journal of Plant Pathology 2000, 106:283-290; , J.1997.Specific probes efficiently distinguishing root-knot nematode species using signature sequence in the ribosomal intergenetic spacer〔J〕Fundamental and Applied Nematology, 20, 619-626; Williamson V.M., et.al., 1997.A to PCRify and Assay Distinguish single juveniles of Meloidogyne hapla and M.chitwoodi〔J〕Journal of Nematology, 1997, 29 (1): 9-15) specific primer; Constructed potato golden nematode (ITS1-Gr, PITSr3) and potato white nematode Specific primers (ITS1-Gp, PITSp4), successfully applied multiplex PCR technology to quickly diagnose potato golden nematode from compound populations (Mulholland V., Carde L., & O.Donnell.1996.Use of the polymerase chain reaction to discriminatepotato cyst nematode at the species level. In: Marshall G. (Ed). Proceedings of diagnostics in Crop Production Symposium. British Crop Production Council, Farnham, UK, pp.247-252; Bulman, S.R. & Marshall, J.W.1997. Differentiation of potaustra cyst nematode (PCN) populations using the polymerase chain reaction (PCR) (J) New Zealand Journal of Crop and Horticultural Science 1997, 25, 123-129.). Constructed soybean cyst nematode-specific primer GlyF1, specific primer GlyF1 and universal primer rDNA2 successfully detected soybean cyst nematode-specific DNA fragment (181bp) (Peng Deliang S.A.Subbotin, Maurice Moens 2001 Application of double PCR technology to detect soybean spore rapidly Cyst nematode. Journal of Shenyang Agricultural University 32(3): 216-217; Subboton S A, Peng, D L and Moens, M.2001A rapid method for the identification of the soybean cyst nematode Heterodera glycines using duplex PCR(J) Nematology, 3(4): 365-371), the sugar beet cyst nematode (H. schachtii) specific primer SHF6 was constructed based on rDNA-ITS. The specific primer SHF6 is combined with the general primer AB28 primer, and the specific fragment is 200bp (Amiri, Subbotin et al., 2002, Identification of the beet cyst nematode Heterodera schachtii by PCR (J) European Journal of Plant Pathology 2002, 108, 497-506. ), the level of diagnosis reaches the level of a single larva.

China has carried out rDNA-ITS analysis of wheat cereal cyst nematode (Peng Deliang, Subbotin.S.A.; Moens, M.2003 ribosomal gene (rDNA) restriction fragment length polymorphism of wheat cereal cyst nematode (Heterodera avenae) Research. Acta Phytopathology, 33(4):323-329), rDNA-ITS-RFLP analysis of soybean cyst nematode populations [Zheng, J.W. (Zheng Jingwu et al.); Peng, D.L.; Moens, M.2000 Stability and variation of its region of rDNA among the inter-populations of soybean cyst nematode, Heterodera glycines, from China. Proceedings of The first Asian conference on Plant Pathology, p198, Beijing, China, August25-28, 2000; Peng Deliang 2001 Toxicity of wheat and soybean cyst nematodes Molecular diagnosis and research on biological control factors of nematode diseases. pp103 (scholar papers)], root-knot nematode rDNA-ITS research [Peng, D.L (Peng Deliang). Subbotin.S.A. & Moens, M.2002.Molecular characteristic of some species of the genus Meloidogyne from China.Nematology, 4(2): 175-176; Liao, J.L (Liao Jinling). Feng, Z.X. & Yang, W.2002. Phylogeny of Meloidogyne spp. Based on rDNA-ITSsequence and fluorescent AFLP. Nematology, 4(2): 173], ribose of pine wood nematode Size and variation analysis of the ITS region of somatic DNA [Liao J.L (Liao Jinling).; Zhang, L.H.; Feng Zh.X. 2000 Identification of Bursaphelenchus xylophilus by PCR Amplification of Ribosomal DNA. Proceedings of The first Asian Conference on Plant Pathology, p202, Beijing , China, August25-28; Zhang Lihai Liao Jinling Feng Zhixin Sequencing and PCR-SSCP analysis of pine xylophilus rDNA. Acta Phytopathology, 2001, 13(1): 84-89; Xu Jianping; Zheng Jingwu; Wang Jianwei PCR Rapid Diagnosis of Pine Xylophilus Journal of Zhejiang Agricultural University 199824(2), 133～134].

At present, the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences has carried out research on the genetic polymorphism of the rDNA-ITS of the stem nematode. The inventor conducted a preliminary study on the rDNA-ITS of the standard sample of the stem nematode harmful to sweet potato from 2001 to 2004, and amplified the rDNA -ITS fragment, which was cloned and sequenced, no matter from the size of the ITS fragment, the distribution pattern of RFLP, or the sequence analysis results, it shows that there are two types of D. nematodes that harm sweet potatoes in my country. Not D. destructor; D. destructor type II is D. destructor. There is no molecular detection method for D. destructor potato in my country so far. The present invention is a rapid and specific molecular detection method for D. destructor potato type II sweet potato nematode.

Contents of the invention

The invention provides the whole sequence of the specific rDNA-ITS of the potato rot nematode through the research on the stem nematode that endangers the sweet potato in my country, and provides its specific detection primer and PCR detection method.

D. destructor potato specific rDNA-ITS is characterized in that it has the nucleotide sequence shown in SEQ NO1.

D. destructor potato specific rDNA-ITS is characterized in that it has a nucleotide sequence as shown in SEQ NO2.

The specific primer SSsj of above-mentioned rDNA-ITS sequence, its nucleotide sequence is as shown in SEQ NO3.

SSsj: 5'-TTGTGTTTGCTGGTGCGCTT-3'.

The rapid PCR detection method of D. destructor potato is characterized in that the PCR reaction system contains the above-mentioned specific primer SSsj and primer AB28, and the nucleotide sequence of the primer AB28 is:

5'-ATATGCTTAAGTTCAGCGGGT-3'.

The above PCR reaction system also contains internal standard primers.

Described internal standard primer is D3A and D3B, and its nucleotide sequence is:

D3A: 5'-GACCCGTCTTGAAACACGGA-3'.

D3B: 5'-TCGGAAGGAACCAGCTACTA-3'.

The rapid PCR detection is a one-step double PCR detection.

The present invention uses the stem nematode disease that harms sweet potato as material, adopts modern molecular biology PCR amplification technology, researches the ribosomal gene (rDNA) of sweet potato stem nematode that harms different regions of my country, and obtains 730bp by amplifying the rDNA-ITS of stem nematode and 900bp fragments, so the D. nematodes that harm sweet potatoes in my country are called type I and type II D. sweet potato, respectively. The samples of D. sweetpotato nematode type II were from Yishui, Shandong and Tongshan, Jiangsu, respectively. The 900bp fragment was cloned and sequenced to obtain the full sequence SEQ NO1 and SEQ NO2 of the rDNA-ITS. Through the comparison of kinship, it was found that the homology with Ditylenchus destructor (Ditylenchus destructor) was very high (96.8% and 92.1%) , so it was confirmed that Ditylenchus type II was Ditylenchus destructor. According to SEQ NO1 and SEQ NO 2, the specific primer (SSsj) of D. potato species harmful to sweet potato was constructed as the upstream primer, and combined with the downstream primer AB28, the molecular biology for rapid and accurate identification and detection of D. potato species harmful to sweet potato was established. According to scientific method and standard PCR technology process, the PCR amplification result of type II sweet potato D. The primers are general primers D3A and D3B for the D3 extension region of the ribosomal DNA28S gene. Using a one-step double PCR method, the PCR amplification results of type II sweet potato D. As a result, only 345bp fragments were amplified, but no 623bp fragments were amplified, and there were no other additional fragments, indicating that the 623bp DNA fragment was a species-specific fragment of D. destructor destructor that harmed sweet potatoes.

The invention provides a fast and accurate molecular detection method for the quarantine inspection of stem nematodes and the identification of stem nematodes in production practice, provides a theoretical reference for making comprehensive management decisions of stem nematode diseases, and is practical for the molecular detection of other important quarantine nematodes entering and leaving the country. Reference meaning.

Description of drawings

Figure 1: The results of rDNA-ITS amplification of the population of D.

M: Standard DNA molecular marker (DL2000), 1: Dm; 2-9: Dd1, Dd2, Dd3, Dd4, Dd5, Dd6, Dd7 and Dd8, 10-11 respectively Dd9 and Dd10; 12 negative controls .

Figure 2: One-step double PCR amplification results of D. destructor-specific primers SSsj and AB28 as well as D3A and D3B. M: standard DNA molecular weight marker (DL2000), lanes 1-9: Dm, Dd1, Dd2, Dd3, Dd4, Dd5, Dd6, Dd7, and Dd8; lanes 10-11, Dd9 and Dd10; lane 12, negative control .

Detailed ways

The present invention will be described in further detail below in conjunction with embodiment.

The material that the experiment of the present invention selects:

Collection of samples of Demia nematode on sweet potato

The samples of stem nematode populations that harm sweet potatoes were collected and collected by our laboratory during the sweet potato harvest season from Lulong, Hebei Funing, Hebei Yixian, Hebei Luanxian, Hebei Luannan and other disease-affected fields, Pingyin County, Shandong Province The sweet potato D. nematode samples were provided by researcher Wang Qingmei from the Institute of Crops, Shandong Academy of Agricultural Sciences; the sweet potato D. nematode samples from Sixian County, Anhui Province and Tongshan County, Jiangsu Province were provided by Associate Researcher Xie Yiping from the Sweet Potato Research Institute of the Chinese Academy of Agricultural Sciences; Daxing District, Beijing The sample was provided by Zhang Jianhua, an agronomist at the Beijing Plant Protection Station; the sample of D. myceliophagus was donated by Dr. Roger Cook of the Welsh Grassland Environmental Research Institute (Table 1).

Table 1 Collection locations and codes of D. nematode populations on sweet potato sample codes Collection location Origin of population latitude and longitude host plant hosts Dd1Dd2Dd3Dd4Dd5Dd6Dd7Dd8Dd9Dd10Dm (D. myceliophagus) Beijing Daxing Daxing Beijing Hebei Lulong Lulong Hebei Hebei Funing Hebei Hebei Yixian Hebei Hebei Luanxian Hebei Hebei Luanxian Hebei Hebei Luannan Luannan Hebei Hebei Pingyin Shandong Pingyin Shandong Anhui Sixian Sixian Anhui Shandong Yishui Shandong Yishui Shandong Jiangsu Tongshan Tongshan Jiangsu UK England East Meridian 116.33 north latitude 39.7 east longitude 118.85 north latitude 39.89 east longitude 119.22 north latitude 39.88 east 115.49 north latitude 39.35 east 118.73 north latitude 39.74 East 118.67 north latitude 39.49 east longitude 36.29 north latitude 33.49 north latitude 35.78 east 34.2 and 117.2 latitude 117.2 latitude 117.2 latitude 117.2 latitude. Sweet Potato Sweet Potato Sweet Potato Sweet Potato Sweet Potato Sweet Potato Sweet Potato Mushroom

Main reagents: Taq DNA polymerase, DNA gel recovery kit, DNA marker were purchased from TaKaRa Company, restriction endonuclease (Biolabs) was purchased from Jingke Hongda Biotechnology Company; primers were synthesized by Shanghai Boya Biotechnology Co., Ltd. ; pGEM-T Easy Vector (Promega, USA) was purchased from Beijing Hualvyuan Biotechnology Company.

Example 1: Amplification of rDNA-ITS of D. destructor potato that damages sweet potatoes

1.1 Extraction of stem nematode DNA

Pick 10 stem nematodes that harm sweet potatoes by hand, put them into a centrifuge tube filled with 14 μl sterilized redistilled water, and freeze them in a -20°C refrigerator for 2 hours. Rotate a glass rod sterilized with 75% alcohol in a centrifuge tube until the ice melts, add 3 μl of 10× PCR buffer, 3 μl of proteinase K solution (600 μg/ml), and freeze at -20°C for at least 1 hour. Take the centrifuge tube out of the refrigerator and incubate at 65°C for 1 hour to degrade deoxyribonucleic acid; then denature proteinase K at 95°C for 10 minutes, centrifuge at 10,000r/min for 1 minute, and take the supernatant DNA suspension in Store at -20°C for later use.

1.2 PCR amplification of rDNA-ITS

Universal primers TW81 and AB28 designed by Joyce et al (1994) were synthesized by Shanghai Boya Biological Co., Ltd. The sequences of TW81 and AB28 are as follows:

TW81 (5'-GTTTCCGTAGGTGAACCTGC-3')

AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3').

Use 50μl PCR reaction system, the ratio is as follows:

10×Buffer (with Mg ²⁺ ) 5μl

10mM dNTP 4μl

ddH ₂ O 37.1 μl

Primer TW81 (1μg/μl) 0.3μl

Primer AB28 (1μg/μl) 0.3μl

Taq enzyme (5U/μl, Takara) 0.3μl

Template DNA 3 μl

Total 50μl

The PCR amplification conditions were 94°C for 4min; 94°C for 1min, 55°C for 2min, 72°C for 2min, 9 cycles; 94°C for 1min; 55°C for 90min; 72°C for 2min; 72°C for 10min; 4°C overnight. After PCR amplification, take 5 μl of the amplified product and add 1 μl of loading buffer for electrophoresis on 1.0% agarose gel, use 1×TAE as the electrophoresis buffer, electrophoresis at 100V for 40 minutes, stain with EB, and observe under purple light take pictures. (figure 1)

The results of PCR amplification showed that D. sweetpotato and D. mycophagous populations in 10 different regions of my country produced single and obvious amplified bands, but the amplified fragments were of different sizes and could be roughly classified into two categories. Eight D. sweetpotato nematode populations (Dd1-Dd8) and mycophagous D. nematode populations (Dm) in Beijing Daxing, Hebei Lulong, Hebei Funing, Hebei Yixian, Hebei Luanxian, Hebei Luannan, Shandong Pingyin, and Anhui Sixian The length of the rDNA-ITS amplified product was about 730bp; while the length of the amplified fragment of D. sweetpotato nematode populations in Yishui, Shandong and Tongshan, Jiangsu was about 920bp, and there was no PCR product in the negative control. Therefore, the stem nematodes that harm sweet potatoes in my country's Beijing Daxing, Shandong Pingyin, Anhui Sixian, Hebei Lulong, Funing, Yixian, Luanxian and Luannan are called type I sweet potato stem nematodes, and Shandong Yishui The stem nematode populations that harm sweet potatoes in Tongshan, Jiangsu and other places are called type II sweet potato stem nematodes.

1.3 Determination of the ITS sequence of D. destructor I TS and the construction of rDNA-based specific marker primers

The 900bp ITS product obtained from D. sweetpotato nematode type II amplified by universal primers TW81 and AB28 was cut from the agarose gel, and the 900bp ITS was recovered and purified using the DNA recovery kit of Dalian Bao Biological Company. According to "Molecular Cloning" CaCl ₂ method to prepare competent cells (Joseph Sambrook, 2001), the purified ITS product was connected to the pGEM-T easy Vector carrier, and after the PCR product was connected to the carrier, it was transformed into E.coli for cloning. The heat shock method was used for transformation, the recombinant plasmid was extracted by alkali method, and the extracted plasmid was identified and detected by restriction endonuclease EcoRJ. The universal primers at both ends of the multi-cloning site of the recombinant plasmid that appeared as a positive clone were sequenced on the insert fragment, and the sequence determination was completed by Beijing Sanbo Polygala Biotechnology Co., Ltd. The sequences were analyzed and compared using Vector NTI 9.0 (IrforrMax Co.) software.

The specific ITS complete sequence of the 900bp D. destructor of potato determined is as follows:

The results of sequence determination showed that the sequences of ITS amplification products of two D. sweetpotato nematode II populations in Shandong Yishui (Dd9) and Jiangsu Tongshan (Dd10) were both 917bp in sequence.

Dd9 1 GTTTCCGTAGGTGAACCTGC TGCCGGATCATTAACGATCATACCAATCCA 50

Dd10 GTTTCCGTAGGTGAACCTGC TGCCGGATCATTAACGATCATACCAATCCA

Dd9 51 CTTTCAGTGGTTATATTAGTCCTCAAAGGTGGCATGCTTCTGCCATGCAG 100

Dd10 CTTTCAGTGGTTATATTAGTCCTCAAAGGTGGCATGCTTCTGCCATGCAG

Dd9 101 GCACAGAGTAGTTGTCCCGCTCTGTATTTGTACTTGCGTTTGGGCTTGCA 150

Dd10 GCACAGAGTAGTTGTCCCGCTCTGTATTTGTACTTGCGTTTGGGCTTGCA

GCT

TG

ACTTGCGCT TGT

CTTGC

CTGTGT

C TGC

CTGT    200Dd9 151 CTTT

GCT

TG

ACTTGCGCT TGT

CTTGC

CTGTGT

C TGC

CTGT 200

TG

ACTTGCGCT TGT

CTTGC

CTGTGT

C

TGC

CTGTDd10 CTTT GCT

TG

ACTTGCGCT TGT

CTTGC

CTGTGT

C

TGC

CTGT

CTTG

GCTGTGT

CTTGCTTTA

AGCTTGCATT

G

GCTTGCATTAG            250Dd9 201 GT

CTTG

GCTGTGT

CTTGCTTTA

AGCTTGCATT

G

GCTTGCATTAG 250

CTTG

GCTGTGT

CTTGCTTTA AGCTTGCATT

G

GCTCGCATTAGDd10 GT

CTTG

GCTGTGT

CTTGCTTTA AGCTTGCATT

G

GCTCGCATTAG

GCATT G

GCTT CATTTGTGCTTG ATTTGCG TTGTGTTTGCT            300Dd9 251 AGCT

GCATT G

GCTT CATTTGTGCTTG ATTTGCG TTGTGTTTGCT 300

GCATT

G

GCTT

CATTTGTGCTTG

ATTTGCG

TTGTGTTTGCTDd10 TGCT

GCATT

G

GCTT

CATTTGTGCTTG

ATTTGCG

TTGTGTTTGCT

Dd9 301 GGTGCGCTTGTGCCTGGCTAATTTGTGGGCGAAAAACGGCTTTGTTGGCC 350

Dd10 GGTGCGCTTGTGCCTGGCTAATTTGTGGGCGAAAAACGGCTTTGTTGGCC

Dd9 351 TCTAAGTTTTTCCTGAGCAGTTGTATGCTTCTTTGTCCGTGGCTGTGATGA 400

Dd10 TCTAAGTTTTTCCTGAGCAGTTGTATGCTTCTTTGTCCGTGGCTGTGATGA

Dd9 401 AGGAAAACGGTACGTGGTTTTCGTAATCGCGAGAGTTAATGAGCACTGGC 450

Dd10 AGGAAAACGGTACGTGGTTTTCGTAATCGCGAGAGTTAATGAGCACTGGC

Dd9 451 TTTGGTGCCGCCAACACAAAACCCCA TTTTACAAATTTTTCAAGAGAAT 500

TTTTACAAATTTTTCAAGAGAATDd10 TTTGGTGCCGCCAACACAAAACCCCA

TTTTACAAATTTTTCAAGAGAAT

Dd9 501 ATTTTTAGTCTTATCGGTGGATCACTCGGCTCGTAGATCGATGAAGAACG 550

Dd10 ATTTTTAGTCTTATCGGTGGATCACTCGGCTCGTAGATCGATGAAGAACG

Dd9 551 CAGCCAACTGCGATAATTAGTGCGAACTGCAGATATTTTGAGCACTAAAG 600

Dd10 CAGCCAACTGCGATAATTAGTGCGAACTGCAGATATTTTGAGCACTAAAG

Dd9 601 TTTCGAATGCACATTGCGCCATTGGATTTTTATCCTTTGGCACGTCTGATT 650

Dd10 TTTCGAATGCACATTGCGCCATTGGATTTTTATCCTTTGGCACGTCTGATT

Dd9 651 CAGGGTCGTAAATACAAAACCCCAAGCTAATGGTGGTGATATGACCTGTG 700

Dd10 CAGGGTCGTAAATACAAAAACCCCAAGCTAATGGTGGTGATATGACCTGTG

Dd9 701 CGGACCGCTGTCTCTTGGCCTAGCACGTGTTTCTTGTGCAGCCTCTTGG 750

Dd10 CGGACCGCTGTCCTCTTTGGCCTAGCACGTGTTTCTTGTGCAGCCTCTTGG

Dd9 751 CCAATGTTGACATCGCTCTCACTCGAGAAAACGCTGTCCAGTGTTTGGTG 800

Dd10 CCAATGTTGACATCGCTCTCACTCGAGAAAACGCTGTCCAGTGTTTGGTG

Dd9 801 ACATTGCTGTAAGTCCTAGCGATTCCTATGGACGTAAGGCTTTGAAGCCA 850

Dd10 ACATTGCTGTAAGTCCTAGCGATTCCTATGGACGTAAGGCTTTGAAGCCA

Dd9 851 AACGCAGAGCAGTCGATTTTTCGACCTGAATCTGACGTGATT ACCCGCTG 900

Dd10 AACGCAGAGCAGTCGATTTTTCGACCTGAATCTGACGTGATT ACCCGCTG

Dd9 901 GCTGAACTTAAGCATAT 917

Dd10 GCTGAACTTAAGCATAT

The sequences of Ditylenchus type II sweet potato nematodes (Dd9 and Dd10) in my country were searched for blast homology on GenBank, and it was found that they had the highest homology with the sequence of Ditylenchus. It was found that the 139-293 position (ITS1) of the ITS sequence in Yishui, Shandong (Dd9) was as high as 96.8% similar to the ITS1 sequence of D. destructor (AF363110) in GeneBank, with only 4 base differences; (Dd10) and its similarity is 92.1%. Comparing the sequences of Dd9 and Dd10, it was found that their ITS sequence similarity was 96.5%. Therefore, it can be confirmed that the Ditylenchus on sweet potatoes in Shandong Yishui (Dd9) and Jiangsu Tongshan (Dd10) is Ditylenchus destructor (Ditylenchus.destructor)

According to the results of the specific ITS sequence analysis and comparison of D. destructor potato, a specific primer for D. destructor potato on sweet potatoes in my country was constructed, named SSsj, and the primer sequence is:

SSsj: 5′-TTGTGTTTGCTGGTGCGCTT-3′

The downstream primer adopts universal primer AB28, and the primer sequence is:

AB28: 5'-ATATGCTTAAGTTCAGCGGGT-3'

It is expected that the amplified region of SSsj and AB28 is 292-917 of the ITS sequence, and the length is 623bp.

Example 2: One-step double PCR detection method of D. destructor specific marker primers and universal primers (D3A and D3B)

The present invention adopts a one-step double PCR method, combines the above-mentioned specific primers SSsj and AB28 to amplify the specific fragment of D. destructor potato, and at the same time, in order to prevent false negative reactions, a general-purpose gene of the D3 region of the ribosomal gene 28S is added to the PCR reaction system. Primers D3A and D3B were used as internal standards, and the one-step double PCR method was used to detect D. destructor potato, which is harmful to sweet potatoes. The one-step double PCR of the same sample was repeated at least four times to confirm the research results and increase reliability.

The sequences of the specific primer SSsj, the universal primer AB28 and the ribosomal DNA28S gene D3 extension region universal primers D3A and D3B of D. destructor are as follows:

Upstream primer SSsj: 5′-TTGTGTTTGCTGGTGCGCTT-3′

Downstream primer AB28: 5'-ATATGCTTAAGTTCAGCGGGT-3'

D3A 5′-GACCCGTCTTGAAACACGGA-3′

D3B 5′-TCGGAAGGAACCAGCTACTA-3′

It is expected that the amplified region of SSsj and AB28 is 292-917 of the ITS sequence, with a length of 623bp; the amplified DNA fragment of D3A and D3B is 345bp,

The one-step dual molecular detection method of D. destructor potato of the present invention contains a 20 μl PCR reaction system, and the proportion is as follows: 10×Buffer (containing Mg ²⁺ ) 2 μl, 10 mM dNTP 1.6 μl, ddH ₂ O 14.4 μl, primer D3A ( 1 μg/l), D3B (1 μg/l), SSsj (1 μg/l), and AB28 (1 μg/l) 0.2 μl each, Taq enzyme (5 U/μl, Takara) 0.2 μl, template DNA 1 μl, add ddH ₂ O to 20 μl .

The amplification program of the PCR reaction was as follows: pre-denaturation at 94°C for 5 min, followed by 1 min at 94°C, 1 min at 55°C, 30 s at 72°C, a total of 35 cycles, and extension at 72°C for 10 rmin. Store at 4°C. Take 5 μl of the amplified product and add 1 liter of loading buffer for electrophoresis on a 1.5% agarose gel, using 1×TAE as the electrophoresis buffer, electrophoresis at 100V for 40 min, stained with EB, observed and photographed under violet light. One-step duplex PCR of the same samples was repeated at least four times to confirm the accuracy of the results.

Using two sets of primers D3A and D3B and specific primers SSsj and AB28, two groups of D. destructor D. destructor affecting sweet potatoes were successfully isolated from Yishui County, Linyi City, Shandong Province (Dd9) and Tongshan County, Xuzhou City, Jiangsu Province (Dd10). Both amplified a specific fragment of 623bp and two obvious DNA fragments of 345bp (lane 10-11 in Figure 2), while in Beijing Daxing, Hebei Lulong, Hebei Funing, Hebei Yixian, Hebei Luanxian, Hebei Type I sweet potato D. nematode populations (Dd1-Dd8) (lanes 2-9 in Figure 2) and mycophagous D. nematode populations (Dm) (1 Only a 345bp fragment is amplified in the swimming lane Dm), and the specific fragment of 623bp is not amplified, and there are no other additional fragments, indicating that the DNA fragment of 623bp is the species-specific fragment of D. The result of co-amplification of D. destructor species specific primer SSsj and universal primer AB28 (Fig. 2).

Attachment: Nucleotide sequence involved in the present invention

The complete sequence of SEQ N01 D. destructor specific rDNA-ITS of potato (sample Dd9)

1 GTTTCCGTAGGTGAACCTGC TGCCGGATCATTAACGATCATACCAATCCA 50

51 CTTTCAGTGGTTATATTAGTCCTCAAAGGTGGCATGCTTCTGCCATGCAG 100

101 GCACACAGAGTAGTTGTCCCGCTCTGTATTTGTACTTGCGTTTGGGCTTGCA 150

151 CTTTGCGCTATGTACTTGCGCTATGTACTTGCTCTGTGTACTTGCTCTGT 200

201 GTACTTGCGCTGTGTACTTGCTTTAGAGCTTGCATTAGTGCTTGCATTAG 250

251 AGCTTGCATTAGAGCTTGCATTTGTGCTTGCATTTGCGCTTGTGTTTGCT 300

301 GGTGCGCTTGTGCCTGGCTAATTTGTGGGCGAAAAACGGCTTTGTTGGCC 350

351 TCTAAGTTTTTCCTGAGCAGTTGTATGCTTCTTTGTCCGTGGCTGTGATGA 400

401 AGGAAAACGGTACGTGGTTTTCGTAATCGCGAGAGTTAATGAGCACTGGC 450

451 TTTGGTGCCGCCAACACAAAACCCCAATTTTACAAATTTTTCAAGAGAAT 500

501 ATTTTTAGTCTTATCGGTGGATCACTCGGCTCGTAGATCGATGAAGAACG 550

551 CAGCCAACTGCGATAATTAGTGCGAACTGCAGATATTTTGAGCACTAAAG 600

601 TTTCGAATGCACATTGCGCCATTGGATTTTTATCCTTTGGCACGTCTGATT 650

651 CAGGGTCGTAAATACAAAACCCCAAGCTAATGGTGGTGATATGACCTGTG 700

701 CGGACCGCTGTCTCTTGGCCTAGCACGTGTTTCTTGTGCAGCCTCTTGG 750

751 CCAATGTTGACATCGCTCTCACTCGAGAAAACGCTGTCCAGTGTTTGGTG 800

801 ACATTGCTGTAAGTCCTAGCGATTCCTATGGACGTAAGGCTTTGAAGCCA 850

851 AACGCAGAGCAGTCGATTTTTCGACCTGAATCTGACGTGATT ACCCGCTG 900

901 GCTGAACTTAAGCATAT 917

The complete sequence of SEQ N02 D. destructor specific rDNA-ITS of potato (sample Dd10)

1 GTTTCCGTAGGTGAACCTGC TGCCGGATCATTAACGATCATACCAATCCA 50

51 CTTTCAGTGGTTATATTAGTCCTCAAAGGTGGCATGCTTCTGCCATGCAG 100

101 GCACACAGAGTAGTTGTCCCGCTCTGTATTTGTACTTGCGTTTGGGCTTGCA 150

151 CTTTCTGCTGTGCACTTGCGCTGTGTGCTTGCGCTGTGTGCCTGCGCTGT 200

201 GTGCTTGTGCTGTGTGCTTGCTTTAAAGCTTGCATTTGCGCTCGCATTAG 250

251 TGCTCGCATTTGTGCTTTCATTTGTGCTTGTATTTGCGGTTGTGTTTGCT 300

301 GGTGCGCTTGTGCCTGGCTAATTTGTGGGCGAAAAACGGCTTTGTTGGCC 350

351 TCTAAGTTTTTCCTGAGCAGTTGTATGCTTCTTTGTCCGTGGCTGTGATGA 400

401 AGGAAAACGGTACGTGGTTTTCGTAATCGCGAGAGTTAATGAGCACTGGC 450

451 TTTGGTGCCGCCAACACAAAACCCCATTTTTACAAATTTTTCAAGAGAAT 500

501 ATTTTTAGTCTTATCGGTGGATCACTCGGCTCGTAGATCGATGAAGAACG 550

551 CAGCCAACTGCGATAATTAGTGCGAACTGCAGATATTTTGAGCACTAAAG 600

601 TTTCGAATGCACATTGCGCCATTGGATTTTTATCCTTTGGCACGTCTGATT 650

651 CAGGGTCGTAAATACAAAACCCCAAGCTAATGGTGGTGATATGACCTGTG 700

701 CGGACCGCTGTCTCTTGGCCTAGCACGTGTTTCTTGTGCAGCCTCTTGG 750

751 CCAATGTTGACATCGCTCTCACTCGAGAAAACGCTGTCCAGTGTTTGGTG 800

801 ACATTGCTGTAAGTCCTAGCGATTCCTATGGACGTAAGGCTTTGAAGCCA 850

851 AACGCAGAGCAGTCGATTTTTCGACCTGAATCTGACGTGATT ACCCGCTG 900

901 GCTGAACTTAAGCATAT 917

SEQ NO3 D. destructor potato specific primer sequence

Upstream primer: SSsj: 5′-TTGTGTTTGCTGGTGCGCTT-3′

Claims (7)

1, potato rot nematode rDNA-ITS is characterized in that having the nucleotide sequence shown in SEQ NO1.

2, potato rot nematode rDNA-ITS is characterized in that having the nucleotide sequence shown in SEQ NO2.

3, the Auele Specific Primer of claim 1 or 2 described potato rot nematode rDNA-ITSs, its nucleotide sequence is shown in SEQNO3.

4, the fast PCR detection method of the rotten Ditylenchus dipsaci of potato is characterized in that containing described Auele Specific Primer of claim 3 and primer AB28 in the PCR reaction system, and the nucleotides sequence of described primer AB28 is classified as: 5 '-ATATGCTTAAGTTCAGCGGGT-3 '.

5, the fast PCR detection method of the rotten Ditylenchus dipsaci of potato according to claim 4 also contains interior label primer in the described PCR reaction system.

6, the fast PCR detection method of the rotten Ditylenchus dipsaci of potato according to claim 5, described interior label primer is D3A and D3B, its nucleotides sequence is classified as:

D3A：5’-GACCCGTCTTGAAACACGGA-3’。

D3B：5’-TCGGAAGGAACCAGCTACTA-3’。

7, it is that an one-step dual PCR detects that the fast PCR detection method of the rotten Ditylenchus dipsaci of potato according to claim 6, described fast PCR detect.

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