Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof

A real-time fluorescence, base ulcer technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of insufficient sensitivity and rapidity in detection, and achieve good application prospects and good repeatability.

Inactive Publication Date: 2010-12-29
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, quarantine ports mostly use traditional morphological methods and conventional PCR techniques to detect the pathogen, which is not sensitive enough and fast enough.

Method used

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  • Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof
  • Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof
  • Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Rape canker canker bacterium real-time fluorescent PCR molecular detection kit

[0029] Composed of:

[0030] Gene Expression Master Mix (product of Applied Biosystems), ddH 2 O,

[0031] MGB probe and primer mix, containing 18 μmol L -1 LMf upstream primer, 18 μmol L -1 LMr downstream primer, 5 μmol L -1 Lmpro probe;

[0032] Positive control is 100ng / μL of rapeseed canker sore DNA solution (bacterial strain: 200782, American Culture Collection Center ATCC);

[0033] Rapeseed canker sore Design and synthesis of MGB probes and primers:

[0034] Through the U.S. National Center for Biotechnology Information (NCBI) ribosomal RNA (rRNA) gene (gene accession numbers: DQ133891, AJ550889, AJ550885) of Rapeseed canker sores and its close species B. After comparing the rRNA genes (NCBI gene accession numbers: FJ172238, AM410082, DQ133893, AJ550871), according to the conserved sequence of the rRNA internal transcription spacer (ITS: internal transcription ...

Embodiment 2

[0040] Example 2 Detection of Rapeseed Canker Bacteria

[0041] 1. The rapeseed canker sore strain samples (seven samples) used for detection are respectively:

[0042] 200782 (American Type Culture Collection ATCC), J-2, J-4, J-9, J-11 (Rape canker canker isolated from Canadian rapeseed), A-7 (from Australian rapeseed canker canker isolated), UK-20 (canker canker in the United Kingdom, donated by the Crop Research Institute of Anhui Academy of Agricultural Sciences).

[0043] 2. Preparation of DNA of Rapeseed canker sore bacteria:

[0044]Get a small amount of mycelium or mycelium block of rape stem base canker sores cultured by each sample, add liquid nitrogen to fully grind them into powder respectively, and use the fungal DNA extraction kit ( plant mini kit, QIAGEN product) to prepare the DNA of each strain.

[0045] 3. Real-time fluorescent PCR reaction

[0046] A total of 20 μL of fluorescent PCR reaction system, including: Gene Expression Master Mix 10μL, MGB p...

Embodiment 3

[0049] Example 3 The specificity test of real-time fluorescent PCR reaction of Rape canker canker bacterium

[0050] 1. Test strains

[0051] Six strains of L.biglobosa, a similar species of rapeseed blackleg (L.biglobosa), among which four strains 2-2, 9-4, 12-2, and 19-3 were donated by Mr. Li Qiangsheng, Institute of Crop Research, Anhui Academy of Agricultural Sciences; CK6 and CK8 are rapeseed black shank (L.biglobosa) isolated from canola; a similar plant stem point fungus (Phoma herbarum) and a seed rot fungus (Phomopsis longicolla); 5 strains of other pathogenic bacteria, respectively CK3 (Leptosphaerulina chartarum), CK7 (Alternaria alternata), CK9 (Alternariaoregonenis), CK12 (Alternaria brassicae) and CK13 (Fusarium proliferatum); in addition, the positive control Canker canker sores (ATCC: 200782); 14 strains in total.

[0052] 2. Preparation of DNA of test strains

[0053] Take a small amount of cultivated mycelia or mycelium blocks of the test strains, add liq...

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Abstract

The invention relates to a real-time fluorescent RCR molecular detection kit for leptosphaeria maculans, which contains a general hybrid reagent for gene expression, ddH2O, a probe and primer mixture and a positive control. The probe and primer mixture contains 5mumol / L of LMpro probe, 18mumol / L of LMf upstream primer and 18mumol / L of LMr downstream primer; and the positive control is 100ng / muL leptosphaeria maculans DNA solution. The kit can directly detect the sick rape seed or plant residues, has the advantages of quickness, sensitivity, accuracy and good repeatability, particularly can be used for detecting a sample with low pathogenic bacteria content, is particularly suitable for quickly detecting the leptosphaeria maculans in seeds, plants and sick residues of cruciferous crops such as imported and exported rape seeds and the like, and has good application prospect.

Description

technical field [0001] The invention relates to a Leptosphaeria maculans (Desm.) Ces. & de Not. real-time fluorescent PCR (or Real-time PCR) molecular detection kit and application thereof, belonging to the field of biotechnology. Background technique [0002] Rapeseed canker Leptosphaeria maculans (Desm.) Ces. & de Not. is a devastating disease in the production process of rapeseed (Brassicanapus Linn). The main natural hosts of the pathogen are cruciferous crops such as rapeseed, cabbage, and Chinese cabbage. The disease can cause the death of seedlings in the field, lodging and premature senescence of adult plants, seriously endangering the yield and quality of rapeseed, a field crop. The disease can occur and prevail under various climatic conditions and different cultivation measures, and the yield loss in a general year is about 10-20%, and the loss in a severe year can reach 30-50%. In Canada, even with strict control measures, the disease causes annual losses of mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 李彬粟寒吴翠萍陈贯源孙民琴吴晶吴新华安榆林
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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