CN114032331B - A specific detection target FPRO_09882 of Fusarium stratum, and its application - Google Patents

A specific detection target FPRO_09882 of Fusarium stratum, and its application Download PDF

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CN114032331B
CN114032331B CN202111517635.5A CN202111517635A CN114032331B CN 114032331 B CN114032331 B CN 114032331B CN 202111517635 A CN202111517635 A CN 202111517635A CN 114032331 B CN114032331 B CN 114032331B
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戴婷婷
徐洁莹
张林巧
焦彬彬
俞萱
吴翠萍
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Nanjing Forestry University
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Abstract

The invention discloses a novel detection target FPRO-09882 of a layered Fusarium (Fusarium proliferatum), and a detection primer, a detection kit and a detection method thereof, wherein the protein sequence of the detection target FPRO-09882 is shown as SEQ ID NO: 1, and the DNA sequence for coding the protein is shown as SEQ ID NO: 2, respectively. Meanwhile, the invention also discloses a specific primer and probe combination of the RPA detection technology for specifically detecting the target FPRO-09882, wherein the forward primer sequence is shown as SEQ ID NO: 3, the reverse primer sequence is shown as SEQ ID NO. 4, and the probe sequence is shown as SEQ ID NO: 5, respectively. The invention discovers a new detection target of the Fusarium laminarization, provides a new detection way for detecting the Fusarium laminarization, and simultaneously, the RPA-LFD detection primer and the probe developed based on the target can realize the specific detection of the Fusarium laminarization and has high sensitivity.

Description

一种层出镰孢菌的特异性检测靶标FPRO_09882及其应用FPRO_09882, a specific detection target of Fusarium stratum, and its application

技术领域technical field

本发明属于基因检测技术领域,具体涉及一种层出镰孢菌(Fusariumproliferatum)的特异性检测靶标FPRO_09882及其应用。The invention belongs to the technical field of gene detection, in particular to a specific detection target FPRO_09882 of Fusarium proliferatum and its application.

背景技术Background technique

层出镰孢菌(F.proliferatum)可侵染水稻、小麦、玉米、芦笋、大蒜、洋葱等作物和杜鹃、油茶、茶花、雪松等木本植物,引起根腐病。层出镰孢菌在PDA平板上可以生长,其形态特征与轮枝镰孢菌F.verticillioides相似,在25℃的条件下培养5d后,菌落呈圆形,常集结成束,中央菌丝体生长茂盛,外围的菌丝体生长相对稀疏,菌丝体由白色发展成了淡黄色,后期中央菌丝体呈现出淡紫色的形态。大型分生孢子呈镰孢形或纺锤形,无色透明,稍微弯曲,并且向两端逐渐变尖,具有2~5个隔膜,大小(24.2~46.7μm)*(1.5~3.7μm),其小型分生孢子在显微镜下观察为长卵形或椭圆形,无色透明,大小(4.4~8.4μm)*(1.2~3.1μm),无隔膜或者有一个隔膜,串生或者假头生,孢子链对生呈“V”型较短,假头生较多,单瓶梗或复瓶梗,复瓶梗较多。不产生无厚垣孢子。在人工培养条件下,该菌生长的最适温度为25℃,最适宜生长的pH值为5-7,分生孢子在相对湿度为100%时,才能很好的萌发,病菌生长致死温度为60℃,10min。病菌生长较好的碳源为可溶性淀粉,氮源为氯化铵。层生镰孢菌的最适生长温度为25℃,35℃以上高温菌丝生长受抑制。该病害分布广、危害重且所致病史症状不易区别,防治困难,是植物生产中最严重的病害之一。当环境条件有利于根腐病发生时,可导致植物大幅度减产或者绝产。层出镰孢菌引起的根腐病是一类土传病害,在国内外均有发生。F. proliferatum can infect rice, wheat, corn, asparagus, garlic, onion and other crops and woody plants such as azalea, camellia, camellia, cedar, etc., causing root rot. Fusarium lamellae can grow on PDA plates, and its morphological characteristics are similar to that of F. verticillioides. After culturing at 25℃ for 5 days, the colonies are round, often aggregated into bundles, and the central hyphae are The body grows vigorously, the peripheral mycelium grows relatively sparsely, the mycelium develops from white to light yellow, and the central mycelium shows a lavender shape in the later stage. The large conidia are fusarium-shaped or spindle-shaped, colorless and transparent, slightly curved, and gradually tapered to both ends, with 2 to 5 septa, the size is (24.2 to 46.7 μm)*(1.5 to 3.7 μm), its Small conidia are observed under microscope as long oval or ellipse, colorless and transparent, size (4.4~8.4μm)*(1.2~3.1μm), without septum or with one septum, bunched or pseudocephalic, spores Chains are short in "V" shape, with more false heads, single or double phlebos, and more phlegm. No chlamydospores are produced. Under artificial culture conditions, the optimum temperature for the growth of the bacteria is 25°C, the optimum pH value for growth is 5-7, the conidia can germinate well when the relative humidity is 100%, and the lethal temperature for the growth of the bacteria is 60℃, 10min. The better carbon source for the growth of bacteria is soluble starch, and the nitrogen source is ammonium chloride. The optimum growth temperature of Fusarium lamelliae was 25℃, and the growth of mycelium at high temperature above 35℃ was inhibited. The disease has a wide distribution, serious damage, and the symptoms of the disease history are difficult to distinguish and difficult to control. It is one of the most serious diseases in plant production. When environmental conditions are favorable for root rot to occur, it can lead to substantial reductions in plant yields or failures. Root rot caused by Fusarium spp. is a type of soil-borne disease that occurs both at home and abroad.

近年来,一些层出镰孢菌的分子检测方法被开发出来。2010年G Mulèe等利用部分钙调素基因序列(calmodulin gene sequences)为从芦笋中分离的层出镰孢菌(F.proliferatum)设计特异性引物,采用普通PCR进行检测。郝芳等人2009年从油茶根腐病中分离得到层出镰孢菌(F.proliferatum),并根据ITS设计出特异性引物,其灵敏度达到1pg,通过巢式pcr扩增后可达到100ag。Riikka Peltomaa等在2016年利用镰孢菌传感器可在食品上检测出镰孢菌,这些传感器是基于特定物种的捕获和检测探针,它们结合到IGS区。寡核苷酸功能化磁性微珠用于捕获目标DNA,然后使用生物素化检测探针和链霉亲和素偶联标签检测目标DNA,它对层出镰孢菌(F.pr oliferatum)的检测灵敏度可达2.0pg。2012年Jonathan等利用Cal基因设计实时荧光定量PCR的探针,灵敏度可达到11pg。2020年YanWang等利用TEF-1α区域作为LAMP检测的目标,通过SYBR Green I目视评估和琼脂糖凝胶电泳检测,目的基因最低检测浓度为10pg。Maria等基于TEF基因设计实时荧光定量PCR的探针,可用在早期侵染水稻种子的层出镰孢菌。这些方法中设计的引物的来源主要都是转录间隔区(ITS)或者TEF基因等保守基因。这些区域或者基因在基因组中都是高拷贝的,所以很容易被检测到。但是越来越多的研究发现,部分疫霉种的ITS序列差异很小,以此为靶标设计的引物难以将不同种区分开来。不同的靶标序列检测的特异性和灵敏度存在一定的差距,因选择的目标靶序列不同、片段大小不同,结果都会有很大差别。因此,发掘特异性好的靶基因是目前层出镰孢菌的检测技术的核心。In recent years, several molecular detection methods for Fusarium sp. have been developed. In 2010, G Mulèe et al. used partial calmodulin gene sequences to design specific primers for F. proliferatum isolated from asparagus, and used ordinary PCR for detection. Hao Fang et al. isolated F. proliferatum from Camellia oleifera root rot in 2009, and designed specific primers based on ITS, with a sensitivity of 1 pg, and 100 ag after amplification by nested PCR. In 2016, Riikka Peltomaa et al. used Fusarium sensors to detect Fusarium spp. in food. These sensors are based on species-specific capture and detection probes that bind to the IGS region. Oligonucleotide-functionalized magnetic microbeads were used to capture target DNA, which was then detected using a biotinylated detection probe and a streptavidin-conjugated tag, which was effective against F. proliferatum. The detection sensitivity can reach 2.0pg. In 2012, Jonathan et al. used Cal gene to design a probe for real-time fluorescence quantitative PCR, with a sensitivity of 11pg. In 2020, Yan Wang et al. used the TEF-1α region as the target of LAMP detection, through SYBR Green I visual assessment and agarose gel electrophoresis detection, and the minimum detection concentration of the target gene was 10pg. Maria et al. designed a real-time fluorescent quantitative PCR probe based on the TEF gene, which can be used for Fusarium stratum stratum that infects rice seeds at an early stage. The primers designed in these methods are mainly from conserved genes such as transcribed spacer (ITS) or TEF gene. These regions or genes are in high copies in the genome, so they can be easily detected. However, more and more studies have found that the ITS sequences of some Phytophthora species differ very little, and primers designed for this purpose are difficult to distinguish between different species. There is a certain gap in the specificity and sensitivity of the detection of different target sequences, and the results will be very different due to different target sequences selected and different fragment sizes. Therefore, the discovery of specific target genes is the core of the current detection technology of Fusarium sp.

发明内容SUMMARY OF THE INVENTION

针对现有技术中层出镰孢菌(F.proliferatum)生物学检测方法所需周期长、检测方法特异性差、灵敏度低、特异性检测靶标较少的问题,本发明提供一种新的层出镰孢菌(F.proliferatum)的检测靶标FPRO_09882及基于新检测靶标其RPA-LFD检测引物组合物。本发明的另一目的是提供上述层出镰孢菌(F.proliferatum)的RPA-LFD检测试剂盒。In view of the problems in the prior art that the biological detection method of F. proliferatum requires a long period of time, the detection method has poor specificity, low sensitivity and fewer specific detection targets, the present invention provides a novel F. proliferatum biological detection method. The detection target FPRO_09882 of F. proliferatum and its RPA-LFD detection primer composition based on the new detection target. Another object of the present invention is to provide the above-mentioned RPA-LFD detection kit for F. proliferatum.

第一方面,本发明提供了一种层出镰孢菌的特异性检测靶标FPRO_09882,所述检测靶标的蛋白序列如SEQ ID NO:1所示:In the first aspect, the present invention provides a specific detection target FPRO_09882 of Fusarium exoplaneta, and the protein sequence of the detection target is shown in SEQ ID NO: 1:

MRRVLGPIAHDKWKNKSTMIGWCLYRAEAGEFTHLLRHWNATEVETAGRLIAWGVPNFVLPALLGLPLAEARTGDDETIYRWVKDEERILEHQYDPFQEGRFNDGGSSADNGYGRSENPYRGRENISGPLGVTHVSARRGNSGSLQQDNRLQEERPGRAGNSTETSHETEAHTLGEIDGLGGFDNFGYREPSNRTRGEGPRSVGNGTKNPYKSNRDGLDR(SEQ ID NO.1)。MRRVLGPIAHDKWKNKSTMIGWCLYRAEAGEFTHLLRHWNATEVETAGRLIAWGVPNFVLPALLGLPLAEARTGDDETIYRWVKDEERILEHQYDPFQEGRFNDGGSSADNGYGRSENPYRGRENISGPLGVTHVSARRGNSGSLQQDNRLQEERPGRAGNSTETSHETEAHTLGEIDGLGGFDNFGYREPSNRTRGEGPRSVGNGTKNPYKSNRDGLDR(SEQ ID NO.1).

另一方面,本发明提供了一种层出镰孢菌的特异性检测靶标FPRO_09882,所述检测靶标的核苷酸列如SEQ ID NO:2所示:On the other hand, the present invention provides a specific detection target FPRO_09882 of Fusarium stratum, the nucleotide sequence of the detection target is shown in SEQ ID NO: 2:

ATGCGACGCGTCCTCGGCCCCATCGCTCACGACAAATGGAAAAACAAGTCGACAATGATAGGCTGGTGTTTATATCGGGCTGAGGCCGGCGAATTTACTCACTTACTAAGGCATTGGAATGCTACAGAGGTGGAAACGGCTGGGAGGTTAATAGCATGGGGAGTACCAAATTTTGTCCTTCCAGCTCTGCTGGGTCTACCGTTGGCAGAGGCTCGAACCGGGGATGATGAGACGATTTACCGATGGGTGAAGGATGAAGAGAGGATACTGGAGCATCAATATGATCCGTTCCAAGAGGGGCGATTCAACGATGGTGGAAGCAGTGCCGACAATGGGTATGGGAGAAGTGAGAATCCGTATAGGGGACGGGAGAACATATCGGGGCCTTTGGGGGTGACTCACGTATCAGCAAGACGTGGGAACTCCGGGAGTTTGCAGCAAGACAATCGTCTTCAGGAGGAGAGACCCGGGAGGGCTGGTAACAGTACCGAGACGTCACATGAGACGGAAGCGCATACCTTGGGGGAGATAGACGGGCTAGGAGGTTTCGACAACTTTGGGTATAGGGAGCCAAGCAATCGTACCCGAGGAGAGGGACCCAGGAGTGTTGGTAACGGTACCAAGAACCCGTACAAGTCGAACCGGGATGGTTTAGACCGTTAA(SEQ ID NO.2)。ATGCGACGCGTCCTCGGCCCCATCGCTCACGACAAATGGAAAAACAAGTCGACAATGATAGGCTGGTGTTTATATCGGGCTGAGGCCGGCGAATTTACTCACTTACTAAGGCATTGGAATGCTACAGAGGTGGAAACGGCTGGGAGGTTAATAGCATGGGGAGTACCAAATTTTGTCCTTCCAGCTCTGCTGGGTCTACCGTTGGCAGAGGCTCGAACCGGGGATGATGAGACGATTTACCGATGGGTGAAGGATGAAGAGAGGATACTGGAGCATCAATATGATCCGTTCCAAGAGGGGCGATTCAACGATGGTGGAAGCAGTGCCGACAATGGGTATGGGAGAAGTGAGAATCCGTATAGGGGACGGGAGAACATATCGGGGCCTTTGGGGGTGACTCACGTATCAGCAAGACGTGGGAACTCCGGGAGTTTGCAGCAAGACAATCGTCTTCAGGAGGAGAGACCCGGGAGGGCTGGTAACAGTACCGAGACGTCACATGAGACGGAAGCGCATACCTTGGGGGAGATAGACGGGCTAGGAGGTTTCGACAACTTTGGGTATAGGGAGCCAAGCAATCGTACCCGAGGAGAGGGACCCAGGAGTGTTGGTAACGGTACCAAGAACCCGTACAAGTCGAACCGGGATGGTTTAGACCGTTAA(SEQ ID NO.2)。

另一方面,本发明还提供了一种检测层出镰孢菌的引物和探针组合,其正向引物FPRO_09882F1序列如SEQ ID NO:3所示,其反向引物FPRO_09882R1序列如SEQ ID NO:4所示,探针序列FPRO_09882P序列如SEQ ID NO:5所示。On the other hand, the present invention also provides a primer and probe combination for detecting Fusarium sp., the sequence of the forward primer FPRO_09882F1 is shown in SEQ ID NO: 3, and the sequence of the reverse primer FPRO_09882R1 is shown in SEQ ID NO: 4, the probe sequence FPRO_09882P sequence is shown in SEQ ID NO:5.

Fpro_09882F1:AACGGCTGGGAGGTTAATAGCATGGGGAGTACCA(SEQ ID NO:3);Fpro_09882F1: AACGGCTGGGAGGTTAATAGCATGGGGAGTACCA (SEQ ID NO: 3);

Fpro_09882R1:ATACGGATTCTCACTTCTCCCATACCCATTGTCG(SEQ ID NO:4);Fpro_09882R1: ATACGGATTCTCACTTCTCCCATACCCATTGTCG (SEQ ID NO: 4);

Fpro_09882P:5'-GCTCGAACCGGGGATGATGAGACGATTTACGATGGGT GAAGGATG-3'(SEQID NO:5),其5'端用FAM标记,3'端用C3Spacer修饰,且在探针的序列中间距5'端的30bp处进行THF修饰。Fpro_09882P: 5'-GCTCGAACCGGGGATGATGAGACGATTTACGATGGGT GAAGGATG-3' (SEQ ID NO: 5), the 5' end was labeled with FAM, the 3' end was modified with C3Spacer, and THF modification was performed at 30 bp from the 5' end in the sequence of the probe.

另一方面,本发明还提供了一种检测层出镰孢菌的试剂盒,至少包括1次用量的含有上述的引物和探针组合的检测溶液。In another aspect, the present invention also provides a kit for detecting Fusarium sp., comprising at least one dose of the detection solution containing the above-mentioned combination of primers and probes.

进一步地,所述试剂盒还包括:装有冻干酶粉的TwistAmp反应单元管、缓冲液Buffer、MgAc、去离子水、HybriDetect assaybuffer、侧流层析试纸条。Further, the kit also includes: a TwistAmp reaction unit tube containing lyophilized enzyme powder, a buffer solution Buffer, MgAc, deionized water, HybriDetect assaybuffer, and lateral flow chromatography test strips.

另一方面,本发明还提供了所述特异性检测靶标FPRO_09882、所述的引物和探针组合以及所述的试剂盒在检测层出镰孢菌中的应用。In another aspect, the present invention also provides the specific detection target FPRO_09882, the primer and probe combination, and the application of the kit in detecting Fusarium sp.

另一方面,本发明还提供了一种层出镰孢菌的检测方法,其特征在于,On the other hand, the present invention also provides a method for detecting Fusarium stratum, characterized in that,

1)提取待测样本DNA;1) Extract the DNA of the sample to be tested;

2)以DNA为模板,利用本发明所述的引物(SEQ ID NO.3、SEQ ID NO.4)和探针(SEQID NO.5)组合或本发明所述的试剂盒进行RPA扩增;2) using DNA as a template, using the combination of primers (SEQ ID NO.3, SEQ ID NO.4) and probes (SEQ ID NO.5) of the present invention or the kit of the present invention to carry out RPA amplification;

3)其中,所述RPA扩增:向装有冻干酶粉的0.2mLTwistAmp反应单元管(TwistAmpnfo kits,Twist)中加入缓冲液Buffer 29.5μL,10μM的上游引物(SEQ ID NO.3)2.1μL,10μM的下游引物(SEQ ID NO.4)2.1μL,探针(SEQ ID NO.5)0.6μL,DNA2.0μL,MgAc 2.5μL加在PCR管盖内部,去离子水补足至50μL;将RPA扩增体系充分混匀,5,000×g离心10s,置于39℃金属浴上反应30min,温育4分钟,将反应管再次混匀,离心3-5s,放入水浴锅39℃继续反应30min。3) wherein, the RPA amplification: add 29.5 μL of buffer buffer to the 0.2 mL TwistAmp reaction unit tube (TwistAmpnfo kits, Twist) containing lyophilized enzyme powder, and 2.1 μL of 10 μM upstream primer (SEQ ID NO.3) , 10 μM downstream primer (SEQ ID NO.4) 2.1 μL, probe (SEQ ID NO.5) 0.6 μL, DNA 2.0 μL, MgAc 2.5 μL inside the PCR tube lid, deionized water to make up to 50 μL; RPA The amplification system was thoroughly mixed, centrifuged at 5,000 × g for 10 s, placed on a metal bath at 39°C for 30 minutes, and incubated for 4 minutes. The reaction tube was mixed again, centrifuged for 3-5 seconds, and placed in a water bath at 39°C for 30 minutes.

4)应用侧流层析试纸条进行RPA扩增产物检测;4) Use lateral flow chromatography test strips to detect RPA amplification products;

取RPA反应产物10μL加入190μL的HybriDetect assay buffer(MileniaBiotec,Giessen,Germany)进行稀释,稀释后取10μL滴在试纸条的HybriDetect 1strip。另一端试纸条垂直插入100μL的HybriDetect assay buffer中,室温放置5min。10 μL of RPA reaction product was added to 190 μL of HybriDetect assay buffer (Milenia Biotec, Giessen, Germany) for dilution, and 10 μL of HybriDetect 1strip was dropped on the test strip after dilution. The other end of the test strip was inserted vertically into 100 μL of HybriDetect assay buffer, and left at room temperature for 5 minutes.

当试纸条出现两条棕色条带,一条位于质控区内,一条位于检测区,则结果为阳性,表明样本中含有层出镰孢菌(F.proliferatum);当试纸条只有质控区出现一条棕色条带,检测区没有条带,则结果是阴性,表明样本中不含有层出镰孢菌(F.proliferatum)。When two brown bands appear on the test strip, one in the quality control area and the other in the detection area, the result is positive, indicating that the sample contains F. proliferatum; when the test strip has only the quality control If a brown band appears in the test area, and there is no band in the detection area, the result is negative, indicating that the sample does not contain F. proliferatum.

本发明的方法与常规PCR相比,检测速度快,不必经过变性、退火、延伸三个步骤,RPA反应的最适温度在25℃-40℃之间,无需变性,在常温下20min左右即可完成反应。这样就实现了恒温扩增,不像PCR法必须要热循环,这样就摆脱了对热循环仪器的依赖,只要有稳定的热源RPA反应就可以发生,极大的扩展了RPA使用的范围,可以真正实现便携式的现场快速核酸检测。PCR反应时间需要1个半小时,而RPA仅需要反应15分钟,大大缩短了检测时间。Compared with conventional PCR, the method of the present invention has faster detection speed and does not need to go through three steps of denaturation, annealing and extension. The optimal temperature of RPA reaction is between 25°C and 40°C, no denaturation is required, and it can be performed at room temperature for about 20 minutes. complete the reaction. In this way, constant temperature amplification is realized. Unlike PCR method, which requires thermal cycling, it gets rid of the dependence on thermal cycling instruments. As long as there is a stable heat source, RPA reaction can occur, which greatly expands the scope of RPA use. Truly realize portable on-site rapid nucleic acid detection. The PCR reaction time takes one and a half hours, while the RPA reaction only takes 15 minutes, which greatly shortens the detection time.

与现有技术相比,本发明的有益效果为:Compared with the prior art, the beneficial effects of the present invention are:

1)本发明提供了一个新的且高信赖度特异性层出镰孢菌分子检测靶标FPRO_09882,为层出镰孢菌的检测提供了一个新的检测途径。1) The present invention provides a new and high-reliability specific FPRO_09882 molecular detection target for Fusarium stratum, which provides a new detection approach for the detection of Fusarium stratum.

2)基于该新靶标建立灵敏、准确、高通量的RPA-LFD检测技术体系,克服了现有技术中层出镰孢菌的生物学检测方法所需周期长、费时费力、繁琐、特异性差的问题及PCR检测技术需要热循环仪器,无法快速检测层出镰孢菌的问题,本发明检测方法在等温条件下,能快速、方便、高效、高特异、高灵敏地检测到层出镰孢菌,不需要复杂仪器,能较好满足对层出镰孢菌的现场检测,对提升层出镰孢菌的快速分子检测研究及其检测时所致病害早期诊断具有重要作用。2) Based on the new target, a sensitive, accurate and high-throughput RPA-LFD detection technology system is established, which overcomes the long cycle, time-consuming, laborious, cumbersome and poor specificity of the biological detection method of Fusarium stratum in the prior art. The problem and PCR detection technology requires thermal cyclers, and cannot quickly detect the problem of Fusarium spp. under isothermal conditions, the detection method of the present invention can detect Fusarium spp. rapidly, conveniently, efficiently, with high specificity and high sensitivity. , does not require complex instruments, can better meet the on-site detection of Fusarium stratiformis, and plays an important role in improving the rapid molecular detection research of Fusarium stratiformis and the early diagnosis of the disease caused by the detection.

3)本发明所提供的检测方法准确性高:本发明根据层出镰孢菌菌检测新靶标FPRO_09882的序列,设计特异性的检测引物和探针组合,LFD侧流层析试纸条检测结果目标条带清析,RPA检测浓度为1pg·μL-1,RPA侧流层析试纸条检测方法的灵敏度要比PCR方法高出100倍。3) The detection method provided by the present invention has high accuracy: the present invention detects the sequence of the new target FPRO_09882 according to Fusarium stratum, design specific detection primers and probe combinations, LFD lateral flow chromatography test strip detection results The target band was cleared, and the RPA detection concentration was 1 pg·μL -1 . The sensitivity of the RPA lateral flow chromatography test strip detection method was 100 times higher than that of the PCR method.

附图说明Description of drawings

为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式描述中所需要使用的附图作简单地介绍。In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings required for the description of the specific embodiments.

图1是基于层出镰孢菌发掘检测靶标FPRO_14873引物的RPA-LFD侧流层析试纸条种间特异性检测结果图;Fig. 1 is a graph showing the results of cross-species specificity detection of the RPA-LFD lateral flow chromatography test strip based on the FPRO_14873 primer of the detection target FPRO_14873;

图2是基于层出镰孢菌(F.proliferatum)新发掘检测靶标FPRO_09882的RPA-LFD侧流层析试纸条在种间特异性检测结果图;Figure 2 is a graph showing the results of interspecies-specific detection of the RPA-LFD lateral flow chromatographic test strip based on the newly discovered detection target FPRO_09882 of F. proliferatum;

图3是基于层出镰孢菌(F.proliferatum)新发掘检测靶标FPRO_09882的RPA-LFD的侧流层析试纸条在属间的特异性检测结果图;Figure 3 is a graph showing the specificity detection results between genera of the lateral flow chromatography test strip of the RPA-LFD of the newly discovered detection target FPRO_09882 based on F. proliferatum;

图4是基于层出镰孢菌新发掘检测新靶标FPRO_09882的设计的特异性引物RPA-LFD的侧流层析试纸条灵敏度检测结果图;4 is a graph showing the sensitivity detection results of lateral flow chromatography test strips based on the design of the specific primer RPA-LFD based on the new discovery and detection of the new target FPRO_09882 by Fusarium stratumbae;

图5是层出镰孢菌新发掘检测新靶标FPRO_09882设计的特异性引物普通PCR灵敏度验证电泳图;Fig. 5 is the electrophoresis image of the general PCR sensitivity verification of specific primers designed by the newly discovered and detected new target FPRO_09882 of Fusarium sp.;

图6是活体组织中层出镰孢菌的RPA-LFD侧流层析试纸条检测结果图。Fig. 6 is a graph showing the detection results of the RPA-LFD lateral flow chromatography test strip of Fusarium stratum in living tissue.

具体实施方式Detailed ways

下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案,因此只是作为示例,而不能以此来限制本发明的保护范围。需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。Embodiments of the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention. It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the usual meanings understood by those skilled in the art to which the present invention belongs.

实施例1Example 1

本研究根据镰孢菌部分属基因组序列(F.proliferatum、Fusariumcircinatum、F.oxysporum、F.graminearum、F.solani、F.fujikuroi、F.odoratissimum、F.verticillioides、F.odoratissimum、F.vanettenii、F.virguliforme、F.clavum、F.mangiferae、F.cerealis、F.redolens、F.sacchari、F.flagelliforme、F.sporotrichioides等共计17种镰孢菌属的全基因组序列分析,通过Blast序列搜索,序列提取、比对与分析,挖掘大规模的基因组数据库,从而发掘疫霉菌的检测靶标。通过全基因组比对,共计获得F.proliferatum特异性检测靶标1000个以上;随机从F.proliferatum1000多个特异性基因中挑选出部分基因作为候选基因,设计了并筛选特异性引物,表1列出其中6个靶标基因(表1)。This study is based on the genome sequences of some Fusarium genus (F.proliferatum, Fusariumcircinatum, F.oxysporum, F.graminearum, F.solani, F.fujikuroi, F.odoratissimum, F.verticillioides, F.odoratissimum, F.vanettenii, F. Whole genome sequence analysis of 17 species of Fusarium such as .virguliforme, F.clavum, F.mangiferae, F.cerealis, F.redolens, F.sacchari, F.flagelliforme, F.sporotrichioides, etc. Extract, compare and analyze, and mine large-scale genome databases to discover the detection targets of Phytophthora. Through the whole genome comparison, more than 1000 specific detection targets of F. proliferatum were obtained in total; more than 1000 specific detection targets of F. proliferatum were randomly selected from Some genes were selected as candidate genes, and specific primers were designed and screened. Table 1 lists 6 target genes (Table 1).

表1 F.proliferatum病原菌6个特异性基因序列表Table 1 Sequence list of six specific genes of F. proliferatum pathogenic bacteria

Figure BDA0003407308740000071
Figure BDA0003407308740000071

Figure BDA0003407308740000081
Figure BDA0003407308740000081

选择与层出镰孢菌不同种(层出镰孢菌(Fusarium proliferatum)、轮枝镰孢菌(F.verticillioides)、松树脂溃疡病原菌(F.circinatum)、藤仓镰孢菌(F.fujikuroi)、茄腐镰孢菌(F.solani)、亚洲镰孢菌(F.asiaticum)、尖镰孢菌(F.oxysporum),以设计的各个靶标引物进行RPA-LFD检测。Select different species from Fusarium proliferatum (Fusarium proliferatum, F. verticillioides, F. circinatum, F. fujikuroi ), F. solani, F. asiaticum, and F. oxysporum, and RPA-LFD detection was performed with each designed target primer.

样品检测:向装有冻干酶粉的0.2mL TwistAmp反应单元管(TwistAm p Basickits,Twist)中加入缓冲液Buffer 29.5μL,10μM的上游引物2.1μL,10μM的下游引物2.1μL,探针0.6μL,DNA 2.0μL,MgAc 2.5μL加在PCR管盖内部,去离子水补足至50μL;将RPA扩增体系充分混匀,5,000×g离心10s,置于39℃金属浴上反应30min,温育4分钟,将反应管再次混匀,离心3-5s,放入水浴锅39℃继续反应30min。Sample detection: Add 29.5 μL of Buffer, 2.1 μL of 10 μM upstream primer, 2.1 μL of 10 μM downstream primer, and 0.6 μL of probe to a 0.2 mL TwistAmp reaction unit tube (TwistAmp Basickits, Twist) containing lyophilized enzyme powder , DNA 2.0 μL, MgAc 2.5 μL was added to the inside of the PCR tube lid, deionized water was added to 50 μL; the RPA amplification system was fully mixed, centrifuged at 5,000 × g for 10 s, placed on a metal bath at 39 °C for 30 min of reaction, and incubated for 4 minutes, the reaction tube was mixed again, centrifuged for 3-5s, and placed in a water bath at 39°C to continue the reaction for 30min.

阴性对照:操作步骤同样品检测,将2.0μL模板DNA改为加入2.0μL灭菌ddH2O。RPA反应结束后,应用侧流层析试纸条进行扩增产物检测。当试纸条出现两条棕色条带,一条位于质控区内(Control line),一条位于检测区(Test line),则结果为阳性,表明样本中含有层出镰孢菌;当试纸条只有质控区出现一条棕色条带,检测区(Test line)没有条带,则结果是阴性,表明样本中不含有层出镰孢菌。Negative control: The operation steps are the same as sample detection, and 2.0 μL of template DNA is changed to add 2.0 μL of sterilized ddH 2 O. After the RPA reaction was completed, the amplification products were detected using lateral flow chromatography test strips. When two brown strips appear on the test strip, one in the control line and the other in the test line, the result is positive, indicating that the sample contains Fusarium spp.; when the test strip Only a brown band appears in the quality control area, and there is no band in the test area (Test line), the result is negative, indicating that the sample does not contain Fusarium stratum.

采用其余5个检测靶标设计的引物和探针,以靶标FPRO_14873为例,其引物序列为:Fpro_14873F:AAACAGAGCGAACAAGTAAGACTAGGGTCGACGA(SEQ ID NO:6);Fpro_14873R:GTGGCACATTTTGCAACTCCCTAAGCGTGTCTTT(SEQ ID NO:7),选择与层出镰孢菌不同种(层出镰孢菌(Fusarium proliferatum)、轮枝镰孢菌(F.verticillioides)、松树脂溃疡病原菌(F.circinatum)、藤仓镰孢菌(F.fujikuroi)、茄腐镰孢菌(F.solani)、亚洲镰孢菌(F.asiaticum)、尖镰孢菌(F.oxysporum),RPA-LFD检测结果显示基于FPRO_14873靶标设计的引物特异性差,结果如图1所示。The primers and probes designed by the other five detection targets are used, taking the target FPRO_14873 as an example, the primer sequences are: Fpro_14873F: AAACAGAGCGAACAAGTAAGACTAGGGTCGACGA (SEQ ID NO: 6); Fpro_14873R: GTGGCACATTTTGCAACTCCCTAAGCGTGTCTTT (SEQ ID NO: 7), select and layer out Different species of Fusarium (Fusarium proliferatum, F. verticillioides, F. circinatum, F. fujikuroi, eggplant rot F. solani, F. asiaticum, and F. oxysporum, the RPA-LFD detection results show that the primers designed based on the FPRO_14873 target have poor specificity, and the results are shown in Figure 1 .

综合分子靶标的检测特异性和灵敏度考虑,筛选获得分子检测靶标FPRO_09882为最终层出镰孢菌的检测新靶标,以及特异性引物及探针组合,其正向引物序列如SEQ IDNO:3所示,反向引物序列如SEQ ID NO:4所示,探针序列如SEQ ID NO:5所示。Considering the detection specificity and sensitivity of molecular targets, the molecular detection target FPRO_09882 was obtained by screening as a new target for the final detection of Fusarium spp., as well as a combination of specific primers and probes. The forward primer sequence is shown in SEQ ID NO: 3 , the reverse primer sequence is shown in SEQ ID NO:4, and the probe sequence is shown in SEQ ID NO:5.

实施例2Example 2

为了验证层出镰孢菌(F.proliferatum)的特异性引物序列,本实施例以层出镰孢菌(F.proliferatum)菌株和病原真菌以及其它卵菌为供试材料(表2),采用CTAB法提取发病组织中层出镰孢菌(F.proliferatum)的DNA。具体方法如下:取少量菌丝粉,加900μL 2%CTAB提取液和90μL 10%SDS,漩涡混匀,于60℃水浴1h,中间每10min上下颠倒几次。12000rpm离心10min,取上清加等体积酚/氯仿/异戊醇(25:24:1),颠倒混匀,12000rpm离心10min;将上清转移至新管,加等体积氯仿,轻轻颠倒混匀,12000rpm离心5min。上清转移至新管中,加2倍体积的无水乙醇和1/10体积的3M NaAc(pH 5.2),-20℃沉淀(>1h)。12000rpm离心10min,倾去上清,沉淀用70%乙醇洗涤两次,室温晾干。加适量灭菌超纯水或TE(pH8.0)溶解沉淀(含20μg/mL RNase),37℃处理1h后,-20℃保存备用。In order to verify the specific primer sequences of F. proliferatum, in this example, F. proliferatum strains and pathogenic fungi and other oomycetes were used as test materials (Table 2). The DNA of F. proliferatum was extracted from the diseased tissue by CTAB method. The specific method is as follows: take a small amount of mycelial powder, add 900 μL of 2% CTAB extract and 90 μL of 10% SDS, vortex and mix, and place in a water bath at 60° C. for 1 h, inverting several times every 10 min in between. Centrifuge at 12000rpm for 10min, take the supernatant and add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), invert and mix, and centrifuge at 12000rpm for 10min; transfer the supernatant to a new tube, add an equal volume of chloroform, and gently mix by inversion Homogenize and centrifuge at 12,000 rpm for 5 min. The supernatant was transferred to a new tube, 2 volumes of absolute ethanol and 1/10 volume of 3M NaAc (pH 5.2) were added, and precipitated at -20°C (>1h). Centrifuge at 12,000 rpm for 10 min, pour off the supernatant, wash the precipitate twice with 70% ethanol, and dry at room temperature. Add an appropriate amount of sterile ultrapure water or TE (pH8.0) to dissolve the precipitate (containing 20 μg/mL RNase), treat at 37°C for 1 h, and store at -20°C for later use.

表2用于层出镰孢菌(F.proliferatum)RPA-LFD检测的真菌和卵菌菌株Table 2 Fungal and oomycete strains for F. proliferatum RPA-LFD detection

Figure BDA0003407308740000101
Figure BDA0003407308740000101

按照实施例1的方法进行检测,检测结果如图2-3所示。The detection is carried out according to the method of Example 1, and the detection results are shown in Figures 2-3.

图2是基于层出镰孢菌(F.proliferatum)新发掘检测新靶标FPRO_09882的RPA-LFD侧流层析试纸条特异性检测结果图;1,2,层出镰孢菌(Fusarium proliferatum);3,松树脂溃疡病原菌(F.circinatum);4,藤仓镰孢菌(F.fujikuroi);5,茄腐镰孢菌(F.solani);6,轮枝镰孢菌(F.verticill ioides);7,亚洲镰孢菌(F.asiaticum);8,N阴性对照。图2中显示1号有2条带,其中一条带为质控线(Control Line),一条线为检测线(TestLine),因此呈阳性,其余只有一条质控线(Control Line)的条带呈阴性,表明样本中含有层出镰孢菌(F.proliferatum);说明新发掘检测靶标FPR O_09882具有种间的特异性。Figure 2 shows the specific detection results of the RPA-LFD lateral flow chromatography test strip based on the new discovery of F. proliferatum to detect the new target FPRO_09882; 1, 2, Fusarium proliferatum 3, Pine resin canker pathogen (F.circinatum); 4, Fusarium fujikura (F.fujikuroi); 5, Fusarium solani (F.solani); 6, Fusarium verticillium (F.verticill) ioides); 7, F. asiaticum; 8, N negative control. Figure 2 shows that No. 1 has 2 bands, one of which is the Control Line and the other is the Test Line, so it is positive, and the rest have only one Control Line. Negative, indicating that the sample contains F. proliferatum; indicating that the newly discovered detection target FPR O_09882 has interspecies specificity.

图3是基于高信赖度特异性分子检测新靶标FPRO_09882的RPA-LFD侧流层析试纸条在属间的特异性检测结果图检测结果图;1:层出镰孢菌(F.proliferatum);2:终极腐霉(Pythium ultimum);3:丁香疫霉(Phytop hthora syringae);4:平头炭疽菌(Colletotrichum truncatum);5:大丽轮枝菌(Verticilium dahliae);6:立枯丝核菌(Rhizoctonia solani);7:稻瘟病菌(Magnaporthe grisea);8:阴性对照;图3中显示第1条有2条带,其中一条带为质控线(Control line),一条线为检测线,因此呈阳性,表明样本中含有层出镰孢菌;其余只有一条质控线(Control line)的条带呈阴性。说明新发掘检测靶标FPRO_09882具有属间的特异性。可见,以实施例1设计的引物进行RPA扩增反应后,LFD侧流层析试纸条检测结果显示2条棕色条带,目标条带清析,能有效检测出层出镰孢菌。而其它真菌和卵菌只有在质控区出现一条棕色条带,阴性对照也只有在质控区出现一条棕色条带。Figure 3 is the specificity detection result of the RPA-LFD lateral flow chromatography test strip based on the high-reliability specific molecular detection of the new target FPRO_09882 between genera; 1: F. proliferatum ; 2: Pythium ultimum; 3: Phytop hthora syringae; 4: Colletotrichum truncatum; 5: Verticilium dahliae; 6: Rhizoctonia solani Rhizoctonia solani; 7: Magnaporthe grisea; 8: Negative control; Figure 3 shows that the first line has 2 bands, one of which is the control line and the other is the detection line , so it was positive, indicating that the sample contained Fusarium stratum stratum; the remaining only one control line was negative. This indicated that the newly discovered detection target FPRO_09882 had intergeneric specificity. It can be seen that after the RPA amplification reaction is carried out with the primers designed in Example 1, the detection result of the LFD lateral flow chromatography test strip shows 2 brown bands, and the target band is clear, which can effectively detect Fusarium stratum. While other fungi and oomycetes only had a brown band in the quality control area, and the negative control only had a brown band in the quality control area.

实施例3Example 3

用同样浓度的层出镰孢菌的标准菌株的基因组DNA作为扩增模板,利用本发明的RPA扩增反应后,LFD侧流层析试纸条检测结果结果如图4所示目标条带清析。由此可见,基于新发掘靶标FPRO_09882,RPA侧流层析试纸条检测方法的检测结果如图4所示,灵敏度为1pg·μL-1,而PCR检测结果如图5所示,其检测浓度为100pg·μL-1时能看到目标条带,说明RPA检测的灵敏度高于PCR。RPA检测时间仅需30min,且不需要PCR仪等昂贵的仪器设备,操作程序简便,更有利在生产中推广应用。Using the genomic DNA of the standard strain of Fusarium layered at the same concentration as the amplification template, after using the RPA amplification reaction of the present invention, the LFD lateral flow chromatography test strip detection results are shown in Figure 4. The target band is clear. analysis. It can be seen that, based on the newly discovered target FPRO_09882, the detection results of the RPA lateral flow chromatography test strip detection method are shown in Figure 4, and the sensitivity is 1pg·μL -1 , while the PCR detection results are shown in Figure 5, the detection concentration When it is 100pg·μL -1 , the target band can be seen, indicating that the sensitivity of RPA detection is higher than that of PCR. The RPA detection time only takes 30 minutes, and does not require expensive equipment such as PCR machines. The operation procedure is simple, and it is more conducive to popularization and application in production.

实施例4Example 4

采用NaOH碱裂解法提取接种层出镰孢菌的发病松针的DNA,将其作为模板用于PCR扩增。取1uL DNA溶液,按实施例3的方法,进行RPA-LFD检测。结果如图6所示,检测结果图中从左边到右边分别为1,层出镰孢菌(F.proliferatum)提取的DNA作为阳性对照;2-4人工接种层出镰孢菌(F.proliferatum)的松针(Cedrus dedara)提取的DNA;5-7人工接种琼脂块的松针(Cedrus deodara)提取的DNA;8松针(Cedrus deodara)提取的DNA;9无菌水(阴性对照)。The DNA of the diseased pine needles of Fusarium sp. from the inoculation layer was extracted by NaOH alkaline lysis method and used as a template for PCR amplification. Take 1 uL of DNA solution and carry out RPA-LFD detection according to the method of Example 3. The results are shown in Figure 6. The test results are 1 from the left to the right, and the DNA extracted from F. proliferatum is used as a positive control; 2-4 are artificial inoculation layers. F. proliferatum ) DNA extracted from pine needles (Cedrus dedara); 5-7 DNA extracted from pine needles (Cedrus deodara) artificially inoculated agar blocks; 8 DNA extracted from pine needles (Cedrus deodara); 9 sterile water (negative control).

RPA扩增反应后,LFD侧流层析试纸条检测结果显示,接种层出镰孢菌的发病松针(2、3、4)能显示2条棕色条带,目标条带清析,能有效检测出层出镰孢菌。而其它真菌和卵菌只有在质控区出现一条棕色条带,阴性对照也只有在质控区出现一条棕色条带。After the RPA amplification reaction, the LFD lateral flow chromatography test strip detection results showed that the diseased pine needles (2, 3, 4) with Fusarium sp. Fusarium sp. stratum was detected. While other fungi and oomycetes only had a brown band in the quality control area, and the negative control only had a brown band in the quality control area.

除非另外具体说明,否则在这些实施例中阐述的数值并不限制本发明的范围。在这里示出和描述的所有示例中,除非另有规定,任何具体值应被解释为仅仅是示例性的,而不是作为限制,因此,示例性实施例的其他示例可以具有不同的值。Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless stated otherwise, any specific value should be construed as merely exemplary and not as limiting, as other examples of exemplary embodiments may have different values.

序列表sequence listing

<110> 南京林业大学<110> Nanjing Forestry University

<120> 一种层出镰孢菌的特异性检测靶标FPRO_09882及其应用<120> A specific detection target FPRO_09882 of Fusarium stratum and its application

<130> 2021<130> 2021

<160> 20<160> 20

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 220<211> 220

<212> PRT<212> PRT

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 1<400> 1

Met Arg Arg Val Leu Gly Pro Ile Ala His Asp Lys Trp Lys Asn LysMet Arg Arg Val Leu Gly Pro Ile Ala His Asp Lys Trp Lys Asn Lys

1 5 10 151 5 10 15

Ser Thr Met Ile Gly Trp Cys Leu Tyr Arg Ala Glu Ala Gly Glu PheSer Thr Met Ile Gly Trp Cys Leu Tyr Arg Ala Glu Ala Gly Glu Phe

20 25 30 20 25 30

Thr His Leu Leu Arg His Trp Asn Ala Thr Glu Val Glu Thr Ala GlyThr His Leu Leu Arg His Trp Asn Ala Thr Glu Val Glu Thr Ala Gly

35 40 45 35 40 45

Arg Leu Ile Ala Trp Gly Val Pro Asn Phe Val Leu Pro Ala Leu LeuArg Leu Ile Ala Trp Gly Val Pro Asn Phe Val Leu Pro Ala Leu Leu

50 55 60 50 55 60

Gly Leu Pro Leu Ala Glu Ala Arg Thr Gly Asp Asp Glu Thr Ile TyrGly Leu Pro Leu Ala Glu Ala Arg Thr Gly Asp Asp Glu Thr Ile Tyr

65 70 75 8065 70 75 80

Arg Trp Val Lys Asp Glu Glu Arg Ile Leu Glu His Gln Tyr Asp ProArg Trp Val Lys Asp Glu Glu Arg Ile Leu Glu His Gln Tyr Asp Pro

85 90 95 85 90 95

Phe Gln Glu Gly Arg Phe Asn Asp Gly Gly Ser Ser Ala Asp Asn GlyPhe Gln Glu Gly Arg Phe Asn Asp Gly Gly Ser Ser Ala Asp Asn Gly

100 105 110 100 105 110

Tyr Gly Arg Ser Glu Asn Pro Tyr Arg Gly Arg Glu Asn Ile Ser GlyTyr Gly Arg Ser Glu Asn Pro Tyr Arg Gly Arg Glu Asn Ile Ser Gly

115 120 125 115 120 125

Pro Leu Gly Val Thr His Val Ser Ala Arg Arg Gly Asn Ser Gly SerPro Leu Gly Val Thr His Val Ser Ala Arg Arg Gly Asn Ser Gly Ser

130 135 140 130 135 140

Leu Gln Gln Asp Asn Arg Leu Gln Glu Glu Arg Pro Gly Arg Ala GlyLeu Gln Gln Asp Asn Arg Leu Gln Glu Glu Arg Pro Gly Arg Ala Gly

145 150 155 160145 150 155 160

Asn Ser Thr Glu Thr Ser His Glu Thr Glu Ala His Thr Leu Gly GluAsn Ser Thr Glu Thr Ser His Glu Thr Glu Ala His Thr Leu Gly Glu

165 170 175 165 170 175

Ile Asp Gly Leu Gly Gly Phe Asp Asn Phe Gly Tyr Arg Glu Pro SerIle Asp Gly Leu Gly Gly Phe Asp Asn Phe Gly Tyr Arg Glu Pro Ser

180 185 190 180 185 190

Asn Arg Thr Arg Gly Glu Gly Pro Arg Ser Val Gly Asn Gly Thr LysAsn Arg Thr Arg Gly Glu Gly Pro Arg Ser Val Gly Asn Gly Thr Lys

195 200 205 195 200 205

Asn Pro Tyr Lys Ser Asn Arg Asp Gly Leu Asp ArgAsn Pro Tyr Lys Ser Asn Arg Asp Gly Leu Asp Arg

210 215 220 210 215 220

<210> 2<210> 2

<211> 663<211> 663

<212> DNA<212> DNA

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 2<400> 2

atgcgacgcg tcctcggccc catcgctcac gacaaatgga aaaacaagtc gacaatgata 60atgcgacgcg tcctcggccc catcgctcac gacaaatgga aaaacaagtc gacaatgata 60

ggctggtgtt tatatcgggc tgaggccggc gaatttactc acttactaag gcattggaat 120ggctggtgtt tatatcgggc tgaggccggc gaatttactc acttactaag gcattggaat 120

gctacagagg tggaaacggc tgggaggtta atagcatggg gagtaccaaa ttttgtcctt 180gctacagagg tggaaacggc tgggaggtta atagcatggg gagtaccaaa ttttgtcctt 180

ccagctctgc tgggtctacc gttggcagag gctcgaaccg gggatgatga gacgatttac 240ccagctctgc tgggtctacc gttggcagag gctcgaaccg gggatgatga gacgatttac 240

cgatgggtga aggatgaaga gaggatactg gagcatcaat atgatccgtt ccaagagggg 300cgatgggtga aggatgaaga gaggatactg gagcatcaat atgatccgtt ccaagagggg 300

cgattcaacg atggtggaag cagtgccgac aatgggtatg ggagaagtga gaatccgtat 360cgattcaacg atggtggaag cagtgccgac aatgggtatg ggagaagtga gaatccgtat 360

aggggacggg agaacatatc ggggcctttg ggggtgactc acgtatcagc aagacgtggg 420aggggacggg agaacatatc ggggcctttg ggggtgactc acgtatcagc aagacgtggg 420

aactccggga gtttgcagca agacaatcgt cttcaggagg agagacccgg gagggctggt 480aactccggga gtttgcagca agacaatcgt cttcaggagg agagacccgg gagggctggt 480

aacagtaccg agacgtcaca tgagacggaa gcgcatacct tgggggagat agacgggcta 540aacagtaccg agacgtcaca tgagacggaa gcgcatacct tgggggagat agacgggcta 540

ggaggtttcg acaactttgg gtatagggag ccaagcaatc gtacccgagg agagggaccc 600ggaggtttcg acaactttgg gtatagggag ccaagcaatc gtacccgagg agagggaccc 600

aggagtgttg gtaacggtac caagaacccg tacaagtcga accgggatgg tttagaccgt 660aggagtgttg gtaacggtac caagaacccg tacaagtcga accgggatgg tttagaccgt 660

taa 663taa 663

<210> 3<210> 3

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 3<400> 3

aacggctggg aggttaatag catggggagt acca 34aacggctggg aggttaatag catggggagt acca 34

<210> 4<210> 4

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 4<400> 4

atacggattc tcacttctcc catacccatt gtcg 34atacggattc tcacttctcc catacccatt gtcg 34

<210> 5<210> 5

<211> 45<211> 45

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 5<400> 5

gctcgaaccg gggatgatga gacgatttac gatgggtgaa ggatg 45gctcgaaccg gggatgatga gacgatttac gatgggtgaa ggatg 45

<210> 6<210> 6

<211> 660<211> 660

<212> DNA<212> DNA

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 6<400> 6

atgagccaag agctaaatga caagcagttt taccaagctt cggccccatt attaacgatc 60atgagccaag agctaaatga caagcagttt taccaagctt cggccccatt attaacgatc 60

gaaaggaaag catttatcaa tgctacatgg aagtcttcca aagccttcaa cgaatttgaa 120gaaaggaaag catttatcaa tgctacatgg aagtcttcca aagccttcaa cgaatttgaa 120

ttgtcaggct actttaggtt ccttgaaaca gagcgaacaa gtaagactag ggtcgacgat 180ttgtcaggct actttaggtt ccttgaaaca gagcgaacaa gtaagactag ggtcgacgat 180

cccgcatact ctcacctttg ttttaaacac attttcgaca ttcaaagtat tctctacgaa 240cccgcatact ctcacctttg ttttaaacac attttcgaca ttcaaagtat tctctacgaa 240

aacctcaatg aacctccgct acgcctacaa gacttagtcc aaagaagctt agtcttgtgg 300aacctcaatg aacctccgct acgcctacaa gacttagtcc aaagaagctt agtcttgtgg 300

gaactagagc tcgaagtcgt tcaggactta atcattctcg ttattaagtt aacatttatg 360gaactagagc tcgaagtcgt tcaggactta atcattctcg ttattaagtt aacatttatg 360

gttcgggccg agttcccaaa ctcctactcg catccttcgc cattccaaat gcagatgcaa 420gttcgggccg agttcccaaa ctcctactcg catccttcgc cattccaaat gcagatgcaa 420

gacaaccaaa gccttaaaga cacgcttagg gagttgcaaa atgtgccacc tttacagaat 480gacaaccaaa gccttaaaga cacgcttagg gagttgcaaa atgtgccacc tttacagaat 480

ttgggtacac aaaatgagct gccgtcgtgg tttaatgtta ttgatctgga gaaaaaggct 540ttgggtacac aaaatgagct gccgtcgtgg tttaatgtta ttgatctgga gaaaaaggct 540

aacctgagaa ttggttggac tgaccatctt gatgaacatc tgactttcca aagtggaact 600aacctgagaa ttggttggac tgaccatctt gatgaacatc tgactttcca aagtggaact 600

cttgtgatat ttcgacatat cgcagtcctc atgtatatga gagaatcagg aatcttgtga 660cttgtgatat ttcgacatat cgcagtcctc atgtatatga gagaatcagg aatcttgtga 660

<210> 7<210> 7

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 7<400> 7

aaacagagcg aacaagtaag actagggtcg acga 34aaacagagcg aacaagtaag actagggtcg acga 34

<210> 8<210> 8

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 8<400> 8

gtggcacatt ttgcaactcc ctaagcgtgt cttt 34gtggcacatt ttgcaactcc ctaagcgtgt cttt 34

<210> 9<210> 9

<211> 660<211> 660

<212> DNA<212> DNA

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 9<400> 9

atgactcgat tcagcaccac ggtagaagat gttcagaaca tcccgcaagg cggtgttagg 60atgactcgat tcagcaccac ggtagaagat gttcagaaca tcccgcaagg cggtgttagg 60

ctactcttac gtagagagaa tgctggagaa acagacactt ggtatgagga ggatttcgat 120ctactcttac gtagagagaa tgctggagaa acagacactt ggtatgagga ggatttcgat 120

catcttgtcg tcgcgacggg ccacaacagt gtcccccgcg tgcccaagat acccggcctc 180catcttgtcg tcgcgacggg ccacaacagt gtccccccgcg tgcccaagat acccggcctc 180

gaggcctgga acggaagcct gcaacatgcc tccacatggc gatcggcaca agagttcaag 240gaggcctgga acggaagcct gcaacatgcc tccacatggc gatcggcaca agagttcaag 240

ggcaagaaaa tcttagtggt cggtaccagt gagagtgcca tcgatctcgt tcttcagtct 300ggcaagaaaa tcttagtggt cggtaccagt gagagtgcca tcgatctcgt tcttcagtct 300

cttcctcatg ccaagggcga tattcatgtg tctcaaagaa aaccgcaccc ccggtatcct 360cttcctcatg ccaagggcga tattcatgtg tctcaaagaa aaccgcaccc ccggtatcct 360

aacgtgtttg atcggcctgg cgtcaaactc gtgaccacga ttgaccattt taccgaagac 420aacgtgtttg atcggcctgg cgtcaaactc gtgaccacga ttgaccattt taccgaagac 420

tctattcatc tagatgacgg ttctgtcttg ggaggcatcg atgtggtagt ttttgcgacg 480tctattcatc tagatgacgg ttctgtcttg ggaggcatcg atgtggtagt ttttgcgacg 480

gggtacttct acacgtaccc cttcctctcg aatgtccgcc cacctgtatc ctgcaggagt 540gggtacttct acacgtaccc cttcctctcg aatgtccgcc cacctgtatc ctgcaggagt 540

ttagtctgtc ctgagagaga aaaacaagta gggtcgccaa tgcgtccaaa tttaacctgc 600ttagtctgtc ctgagagaga aaaacaagta gggtcgccaa tgcgtccaaa tttaacctgc 600

aaagcaacaa caaagggaaa ctggagctat ttcttaatcc gatggggttc aatcaactag 660aaagcaacaa caaagggaaa ctggagctat ttcttaatcc gatggggttc aatcaactag 660

<210> 10<210> 10

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 10<400> 10

caacagtgtc ccccgcgtgc ccaagatacc cggc 34caacagtgtc ccccgcgtgc ccaagatacc cggc 34

<210> 11<210> 11

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 11<400> 11

cacatcgatg cctcccaaga cagaaccgtc atct 34cacatcgatg cctcccaaga cagaaccgtc atct 34

<210> 12<210> 12

<211> 651<211> 651

<212> DNA<212> DNA

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 12<400> 12

atgtttgtat cagaaatcat cagccctctc ctcctcgtcg ccagcgccgc actggcggct 60atgtttgtat cagaaatcat cagccctctc ctcctcgtcg ccagcgccgc actggcggct 60

gccaatcctc agccctcctt tgcggctgtt gtggctcccc gtctgcaggt ttccgtcgaa 120gccaatcctc agccctcctt tgcggctgtt gtggctcccc gtctgcaggt ttccgtcgaa 120

gcagtcgacg aactcctgat gagcaacact actgagcatc tcacaagcat tgatatcgcc 180gcagtcgacg aactcctgat gagcaacact actgagcatc tcacaagcat tgatatcgcc 180

aaatgggctc ctgctctcgg gcagccagtc gaggttctca ccagcttgga ctctgaaacc 240aaatgggctc ctgctctcgg gcagccagtc gaggttctca ccagcttgga ctctgaaacc 240

aagttcaagg tgttctattt cctccaccaa gccatttccg aggggcctgg atcgctttct 300aagttcaagg tgttctattt cctccaccaa gccatttccg aggggcctgg atcgctttct 300

aagcgtgacc aggcgactgc tgataaagat gccgccatca tcagggagaa ggcagaatcc 360aagcgtgacc aggcgactgc tgataaagat gccgccatca tcagggagaa ggcagaatcc 360

taccaaagca cagtcacagt tgaggcagcg gaagatgagc ttcgttgcca gaataccgct 420taccaaagca cagtcacagt tgaggcagcg gaagatgagc ttcgttgcca gaataccgct 420

tcttgtgttc tctgtatcgg tgcagctgca actaccgcgg gaggtctcat ttcttcatgc 480tcttgtgttc tctgtatcgg tgcagctgca actaccgcgg gaggtctcat ttcttcatgc 480

tctgcagttg cactacgagc caacaacctt cgtgttgcag gaaacgctag cccaacagct 540tctgcagttg cactacgagc caacaacctt cgtgttgcag gaaacgctag cccaacagct 540

gccgaggtta gcggtgctgt cattatcgcg gagctcttag catgcgccgc caagcctctt 600gccgaggtta gcggtgctgt cattatcgcg gagctcttag catgcgccgc caagcctctt 600

accggtttcc tcgttgctac tggtgtctgc ctcaaggtta caggccatta a 651accggtttcc tcgttgctac tggtgtctgc ctcaaggtta caggccatta a 651

<210> 13<210> 13

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 13<400> 13

acactactga gcatctcaca agcattgata tcgc 34acactactga gcatctcaca agcattgata tcgc 34

<210> 14<210> 14

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 14<400> 14

gacctcccgc ggtagttgca gctgcaccga taca 34gacctcccgc ggtagttgca gctgcaccga taca 34

<210> 15<210> 15

<211> 507<211> 507

<212> DNA<212> DNA

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 15<400> 15

atggcaactc cgatttccca agaaccctct gggccgtgtt cagaacaaga acaaatgaat 60atggcaactc cgatttccca agaaccctct gggccgtgtt cagaacaaga acaaatgaat 60

gaaatgtcta gaaatttcta ttacgttaga aagagagaag tccgtatgtt tccaggcgca 120gaaatgtcta gaaatttcta ttacgttaga aagagagaag tccgtatgtt tccaggcgca 120

acggctttgc taaaaatgtc gattcagaag tacatggcga agtacgaggt agaatttatg 180acggctttgc taaaaatgtc gattcagaag tacatggcga agtacgaggt agaatttatg 180

gatgacgacc aaaagcttcg cgttttgctg cctctggatg tgaataggga ggactctgat 240gatgacgacc aaaagcttcg cgttttgctg cctctggatg tgaataggga ggactctgat 240

aagaaattcc aactgctgat ggaaatgccc gataatatga agaaggcgaa gcttttgacc 300aagaaattcc aactgctgat ggaaatgccc gataatatga agaaggcgaa gcttttgacc 300

ttctttcgtg tcgatacgat caaggaattc gaaaaacatg tccagatgac agagatctct 360ttctttcgtg tcgatacgat caaggaattc gaaaaacatg tccagatgac agagatctct 360

atcttaggcc tcgaaaggag aaatacctgc acaagtcgga gaggcaggac ctcaaagatc 420atcttaggcc tcgaaaggag aaatacctgc acaagtcgga gaggcaggac ctcaaagatc 420

aacgagattt ctagcaatac acaaggcaga actggcaaaa cagaaacaaa ttcagggaga 480aacgagattt ctagcaatac acaaggcaga actggcaaaa cagaaacaaa ttcagggaga 480

atagaagagc tgagactcag gagctag 507atagaagagc tgagactcag gagctag 507

<210> 16<210> 16

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 16<400> 16

aatttctatt acgttagaaa gagagaagtc cgta 34aatttctatt acgttagaaa gagagaagtc cgta 34

<210> 17<210> 17

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 17<400> 17

gatcgtatcg acacgaaaga aggtcaaaag cttc 34gatcgtatcg acacgaaaga aggtcaaaag cttc 34

<210> 18<210> 18

<211> 558<211> 558

<212> DNA<212> DNA

<213> 层出镰孢菌(Fusarium proliferatum)<213> Fusarium proliferatum

<400> 18<400> 18

atgcctcgta attcgtctcg cagaaagtct cgccgcagcg agaggtctgc ttcccagtct 60atgcctcgta attcgtctcg cagaaagtct cgccgcagcg agaggtctgc ttcccagtct 60

acacctgagt ctacatcccc atctgcaacc ccgcctactt cacaggattc cccgcgccct 120acacctgagt ctacatcccc atctgcaacc ccgcctactt cacaggattc cccgcgccct 120

cggtcttcga gaccgaccac tcccggctct tcaggccctc agtcttcgag accgaccact 180cggtcttcga gaccgaccac tcccggctct tcaggccctc agtcttcgag accgaccact 180

cccggctctt caggccctca gtctccctgg tcaagcggct ttgaggaaca gggtgtaact 240cccggctctt caggccctca gtctccctgg tcaagcggct ttgaggaaca gggtgtaact 240

ggtctgccca gtcctccagc atcacaatct ccgtctcttg gaacctcgag ccctgctaat 300ggtctgccca gtcctccagc atcacaatct ccgtctcttg gaacctcgag ccctgctaat 300

gtcgctctat catcctctgg aatctcaaca ccatcaattg aggaacatga accttatcgc 360gtcgctctat catcctctgg aatctcaaca ccatcaattg aggaacatga accttatcgc 360

tccgccactc cgatagagga tatgactgca cccccatcta cgactgctag tcagtcgtcg 420tccgccactc cgatagagga tatgactgca cccccatcta cgactgctag tcagtcgtcg 420

gccaaatcta ttaaggctga ccctttggag gatgaaactc tacccaacga tcaggataca 480gccaaatcta ttaaggctga ccctttggag gatgaaactc tacccaacga tcaggataca 480

ggctacgtga tcgagacaat ttaccttctc cattccaaag ccatctggag ttacatccag 540ggctacgtga tcgagacaat ttaccttctc cattccaaag ccatctggag ttacatccag 540

ttcatacttg ggaactag 558ttcatacttg ggaactag 558

<210> 19<210> 19

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 19<400> 19

gctcttcagg ccctcagtct tcgagaccga ccac 34gctcttcagg ccctcagtct tcgagaccga ccac 34

<210> 20<210> 20

<211> 34<211> 34

<212> DNA<212> DNA

<213> 人工序列(artificial sequence)<213> Artificial sequence

<400> 20<400> 20

agatgggggt gcagtcatat cctctatcgg agtg 34agatgggggt gcagtcatat cctctatcgg agtg 34

Claims (5)

1.层出镰孢菌FPRO_09882作为特异性检测靶标在非疾病的诊断和治疗目的的检测层出镰孢菌中的应用,其特征在于,所述FPRO_09882的氨基酸序列如SEQ ID NO:1所示,所述FPRO_09882的核苷酸序列如SEQ ID NO:2所示。1. the application of Fusarium sp. FPRO_09882 as a specific detection target in the detection of Fusarium sp. of non-disease diagnosis and therapeutic purpose, it is characterized in that, the amino acid sequence of described FPRO_09882 is as shown in SEQ ID NO: 1 , the nucleotide sequence of the FPRO_09882 is shown in SEQ ID NO:2. 2.层出镰孢菌FPRO_09882的检测试剂在制备检测层出镰孢菌试剂盒中的应用,其特征在于,所述检测试剂包括引物和探针组合,所述引物包括正向引物和反向引物,所述正向引物序列如SEQ ID NO:3所示,所述反向引物序列如SEQ ID NO:4所示,所述探针序列如SEQID NO:5所示。2. the application of the detection reagent of layering FPRO_09882 in the preparation detection layer of Fusarium spp. kit, is characterized in that, described detection reagent comprises primer and probe combination, and described primer comprises forward primer and reverse Primers, the forward primer sequence is shown in SEQ ID NO: 3, the reverse primer sequence is shown in SEQ ID NO: 4, and the probe sequence is shown in SEQ ID NO: 5. 3.根据权利要求2所述的应用,其特征在于,所述试剂盒至少包括1次用量的所述引物和探针组合。3. The application according to claim 2, wherein the kit comprises at least one dose of the primer and probe combination. 4.根据权利要求3所述的应用,其特征在于,所述试剂盒还包括:装有冻干酶粉的TwistAmp 反应单元管、缓冲液Buffer、MgAc、去离子水、HybriDetect assay buffer、侧流层析试纸条。4. application according to claim 3, is characterized in that, described test kit also comprises: TwistAmp reaction unit tube containing lyophilized enzyme powder, buffer solution Buffer, MgAc, deionized water, HybriDetect assay buffer, side flow Chromatography test strips. 5.一种非疾病的诊断和治疗目的的层出镰孢菌的检测方法,其特征在于,5. the detection method of the Fusarium stratumella of a kind of non-disease diagnosis and therapeutic purpose, is characterized in that, 1)提取待测样本DNA;1) Extract the DNA of the sample to be tested; 2)以DNA为模板,利用权利要求2中所述的引物和探针组合或试剂盒进行RPA扩增;2) using DNA as a template, using the primer and probe combination or kit described in claim 2 to carry out RPA amplification; 3)应用侧流层析试纸条进行RPA扩增产物检测;当试纸条出现两条棕色条带,一条位于质控区内,一条位于检测区,则结果为阳性,表明样本中含有层出镰孢菌;当试纸条只有质控区出现一条棕色条带,检测区没有条带,则结果是阴性,表明样本中不含有层出镰孢菌。3) Use lateral flow chromatography test strips to detect RPA amplification products; when two brown bands appear on the test strip, one in the quality control area and the other in the detection area, the result is positive, indicating that the sample contains layers Fusarium spores; when the test strip only has a brown band in the quality control area, and there is no band in the detection area, the result is negative, indicating that the sample does not contain Fusarium spp.
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