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LAMP primer and kit for detecting Neofusicoccum algeriense

A detection kit and detection reagent technology, applied in DNA/RNA fragments, recombinant DNA technology, microorganisms, etc., can solve the problems of long reaction time, unfavorable grass-roots promotion, and weak prevention effect, and achieve high sensitivity and repeatability, Ease of popularization and use, avoiding the effect of instrument investment

Active Publication Date: 2020-04-21
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, timely monitoring of the initial infection source of this type of pathogen is of great significance for the prevention and control of the disease. Traditional disease prevention and control strategies mainly rely on control measures such as species, cultivation, chemical control, and ecological regulation. The measures are mainly implemented when the disease breaks out or even causes obvious damage, and neglects the early comprehensive prevention and control and efficient control measures for the initial infection source, so it is half the effort with half the effort and little control effect, and it is difficult to control the occurrence and epidemic of the disease in the end
[0003] Ordinary PCR technology requires precision variable temperature equipment and advanced and complex analytical instruments, or requires relatively high proficiency and professional level of operators, and the reaction time is long, which is not conducive to grassroots promotion
So far, there are no related reports on the detection of blueberry stem canker pathogen (Neofusicoccum algeriense) using LAMP technology

Method used

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  • LAMP primer and kit for detecting Neofusicoccum algeriense
  • LAMP primer and kit for detecting Neofusicoccum algeriense
  • LAMP primer and kit for detecting Neofusicoccum algeriense

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Design and synthesis of LAMP primers for detecting blueberry stem canker pathogen (Neofusicoccum algeriense)

[0044] For Neofusicoccum, the translation elongation factors (elongation factors, EF) gene has a higher mutation rate, so the EF gene was selected for species-level identification and molecular systematic research.

[0045] According to the gene sequence of the EF-1α region of blueberry stem canker (Neofusicoccum algeriense) ( figure 1 ), respectively designed three sets of LAMP primers for detecting Neofusicoccum algeriense, including:

[0046] Primer set 1:

[0047] Outer forward primer F3: 5'-GCTTGCAAGTTCTCTGAGCT-3';

[0048] Outer reverse primer B3: 5'-TTGACGCTGTGGAAAAGAGT-3';

[0049] Inner forward primer FIP: 5'-GCGGCATCACCAGACTTGATGAGAGAAGATCGACCGCCGT-3';

[0050] Internal reverse primer BIP: 5'-CATGTGCGTTGAGGCTTTTCACCAGGTTCAAGGGAGGGACAT-3'.

[0051] Primer set 2:

[0052] Outer forward primer F3: 5'-AAAGTTTTTCCTTCCGCTGC-3';

[0053] Out...

Embodiment 2

[0062] Example 2 Specificity analysis of LAMP primers for detection of Neofusicoccum algeriense

[0063] 1.1 Reagents and equipment: LAMP-PCR kit was purchased from Guangzhou Huafeng Company.

[0064] 1.2 Sample source

[0065] Neofusicoccum algeriense, Neofusicoccum illicii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, Lasiodiplodia chinensis, Alternaria alternata, etc. (Table 1) used in this example are preserved in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. These strains are available to the public without depositing them.

[0066] The gene sequence alignment results of the EF-1α region related to fungi in Table 1 can be found in figure 1 .

[0067] Table 1 Sources of samples used for LAMP-PCR detection

[0068]

[0069] 1.3 DNA extraction

[0070] DNA from plant tissues was extracted using the CTAB Plant Genomic DNA Rapid Extraction Kit (Beijing Aidelai Biotechnology Co., Ltd.), and DNA ...

Embodiment 3

[0082] Example 3 Sensitivity analysis of detection of Neofusicoccum algeriense by LAMP primer set

[0083] 1.1 DNA sample concentration:

[0084] The DNA concentration of the Neofusicoccumalgeriense sample extracted in Example 2 was detected by NanoDrop (Thermo Fisher Scientific), and it was 9.8 μg / ml.

[0085] 1.2 Sensitivity detection of LAMP primer set:

[0086] Carry out 10 times serial dilution of DNA sample, take 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 Double-diluted DNA samples were subjected to LAMP constant temperature amplification reaction. Reaction system and reaction condition are with embodiment 2.

[0087] 1.3 Results:

[0088] figure 2 K is the visual effect of the constant temperature amplification reaction system of LAMP primer group 3, and reaction tubes 1-8 are the neofusicoccum algeriense bacteria from 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 For the diluted sample, the reaction system of reaction tube 1-7 shows fluorescen...

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Abstract

The invention provides a set of LAMP primers and a kit for detecting pathogenic bacteria of blueberry stem canker Neofusicoccum algeriense. The LAMP primers comprise outer side forward and reverse primers and inner side forward and reverse primers, and nucleotide sequences of the outer side forward and reverse primers and the inner side forward and reverse primers are shown as SEQ ID NO: 1-4. According to the invention, the LAMP primer isothermal amplification technology is used for rapid detection of Neofusicoccum algeriense pathogens, and the pathogens can be accurately detected from complexpathogen environments in diseased plant tissues and nursery stocks. The specificity, the sensitivity and the repeatability of the method are higher than those of a conventional PCR method, and the method has important significance in the aspects of early warning of Neofusicoccum algeriense, pathogen monitoring of an epidemic area and the like; meanwhile, high instrument investment can be avoided,and primary popularization and application are facilitated.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a LAMP primer and a kit for detecting Neofusicoccumalgeriense. Background technique [0002] Blueberry Stem Blight, which is caused by Botryosphaericeae infection, has been reported in major blueberry producing areas in my country, and has seriously jeopardized the healthy development of China's blueberry industry. The fungus infects blueberry plants from wounds or natural orifices, leading to the death of the cortex and phloem. In the field, blueberry branches wither, xylem necrosis, and plant death are caused. At present, there are many species of Botryosphaeria cortices, Botryosphaeria dothidea, Fusicoccum aesculin, Lasiodiplodia chinesis, Lasiodiplodia theobromae, Lasiodiplodia vaccinii, Neofusicoccum algeriense, Neofusiccum arbuti, Neofusiccum austral, Neofusvusbiricoccum paricoccum) Among them, Neofusicoccum algeriense is a strong pathogen, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2563/107C12Q2565/125C12Q2565/113
Inventor 张英王宇
Owner BEIJING FORESTRY UNIVERSITY