CN105886619B - A method for identifying ergot bacteria in food - Google Patents

Abstract

The invention provides a kit for detecting ergot, which comprises a primer, Bst DNA polymerase, reaction buffer solution and nucleic acid dye. The invention also provides an LAMP method for detecting the ergot bacteria. The LAMP kit or the LAMP method can be used for quickly and accurately identifying and detecting the ergot, has the advantages of high specificity, short time consumption, high sensitivity, simple and convenient identification and the like, and is suitable for laboratory or field inspection.

Description

A method for identifying ergot bacteria in food

technical field

The invention relates to the field of microorganism detection, in particular to a kit and a detection method for detecting ergot bacteria.

Background technique

Ergot fungus (Claviceps purpurea) belongs to the family Ergotaceae and the genus Ergot, and is a kind of fungus mainly taking grass weeds and grain plants as parasitic hosts. It parasitizes in the ovary of the host, and the infected ovary does not form seeds but hard sclerotia. The structure of the sclerotium is relatively dense, and there is a layer of horn-like shell on the outside, hence the name "Ergots" (Alderman, 1999). Ergot fungus grows vigorously under high humidity and high water conditions, and it is easy to infect plants. Most ergot pathogens have 1-4 times more ergot than the seeds of their host plants. The diseases caused by the ergot pathogen and its sclerotia on the host plant are generally called "ergot diseases". Ergot disease is an important disease of wheat and gramineous forages. It not only greatly reduces the yield of wheat, but also the alkaloids in ergot are toxic to humans and animals. In history, poisoning incidents caused by eating grains containing ergot by mistake occurred from time to time , such as livestock eating ergot-containing forage poisoning and death, and people who ingest too much ergot-containing grain food will also experience abortion or even death. Therefore, it is necessary to develop a kit or method suitable for rapid detection of ergot bacteria in the field or in the laboratory.

At present, molecular biology detection has gradually replaced traditional morphological identification in the field of microbial detection due to its rapid and sensitive characteristics, and has become a popular research direction. Loop-mediated isothermal amplification of DNA (LAMP) is a new nucleic acid amplification technology established by Japanese scholar Notomi in 2000. This technology designs 4 primers for 6 regions of the target gene, and uses a DNA polymerase with strand displacement activity to rapidly and highly specifically amplify the target gene under constant temperature conditions. The product is a dumbbell-shaped DNA with stem-loop structures at both ends. molecular. Compared with the PCR method for routine detection in laboratories, LAMP technology has the advantages of simple operation, high sensitivity, and strong specificity, and is widely used in the detection of various pathogens in food, animal and plant inspection and quarantine.

Contents of the invention

The object of the present invention is to provide a LAMP kit and a LAMP method for detecting ergot bacteria.

In order to realize the purpose of the present invention, in one aspect, the present invention provides a kind of LAMP test kit for detecting ergot bacteria, it comprises primer, reaction buffer solution, Bst DNA polymerase and nucleic acid dye, wherein said primer is as follows :

Outer primer F3: CGGACACTCAAGATCGACC (SEQ ID NO: 1)

Outer primer B3: CGATATCGGGCCTTGTGAAT (SEQ ID NO: 2)

Internal primer FIP: TGGGGTTCGTTTCGGCTTGAA-GACAAGCGTGCTTGACCAAT (SEQ ID NO: 3)

Internal primer BIP: GAGAATCTGAGGCCGGCTACTG-TCCTTCCTATGCCCTGCT (SEQ ID NO: 4).

Preferably, the reaction buffer consists of 2 mM dNTPs, 10×ThermoPol reaction buffer, 0.6 mM betaine and 6 mM Mg ²⁺ .

Preferably, the nucleic acid dye is 1000×SYBR Green I.

Preferably, the kit of the present invention further includes DNA extraction reagents, positive controls and negative controls.

Preferably, the DNA extraction reagent is such as CTAB extraction buffer.

In another aspect, the present invention provides the use of primers in preparing a LAMP kit for detecting ergot bacteria. The primers are shown in the sequence of SEQ ID NOs: 1-4.

In another aspect, the present invention also provides a LAMP method for detecting ergot bacteria, wherein the kit of the present invention is used, and the method comprises the following steps:

(1) Add CTAB extraction buffer to the sample to be tested, and extract DNA according to the conventional CTAB method;

(2) Prepare a LAMP reaction system in a PCR tube, including 0.2 μmol/μl outer primer F3 1 μl, 0.2 μmol/μl outer primer B3 1 μl, 1.2 μmol/μl inner primer FIP 1 μl, 1.2 μmol/μl inner primer BIP 1μl, reaction buffer 2.5μl, 8U/μl Bst DNA polymerase 1μl, template DNA 2μl, add water to make up to 25μl, and use ergot genome DNA as positive control, 100mM Tris-HCl pH 8.0 and 50mM EDTA as negative control;

(3) React the PCR tube in step (2) at a constant temperature of 63°C for 60min;

(4) Analyze and judge the result of the reaction product: add 1 μl of nucleic acid dye SYBRGreen I to the reaction product obtained in (3), if the color of the reaction solution is orange, it means that the result is negative, and the food does not contain ergot bacteria, if the color of the reaction solution is green , indicating a positive result.

The inventors of the present invention designed 4 primers for the 6 regions of the ergot sequence, which have high sensitivity and good specificity. The LAMP kit and method for detecting ergot bacteria of the present invention can accurately identify ergot bacteria in food, grains, and fields, and the false positive rate is low, fast and efficient, and the result can be judged by observing the color change with the naked eye, without electrophoresis and ultraviolet observation and other steps, low cost, simple operation, easy identification, suitable for basic laboratory and on-site detection, and worthy of popularization and application.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that various modifications can be made without departing from the spirit of the invention, and these modifications will be included within the scope of the invention.

The preparation of embodiment 1 kit of the present invention

1.1 Reagents

Primers were synthesized by Tiangen Biochemical Technology (Beijing) Co., Ltd.; Bst DNA polymerase and 10×ThermoPol reaction buffer were purchased from Takara; SYBR Green I was purchased from Invitrogen; other PCR reagents and reagents required for preparing CTAB extraction buffer were purchased from From Sigma.

1.2 Preparation of the kit:

The following reagents were obtained to prepare the kit of the invention:

CTAB extraction buffer: prepared according to the following formula: 100mM Tris-HCl pH 8.0, 50mM EDTA, 1M NaCl, 1% (v/v) β-mercaptoethanol, 2% CTAB, adjust the pH value to 7.2;

Reaction buffer: prepared according to the following formula: 2mM dNTP, 10×ThermoPol reaction buffer, 0.6mM betaine, 6mM MgSO ₄ ;

Primer: outer primer F3, its nucleotide sequence is as shown in SEQ ID NO:1; Outer primer B3, its nucleotide sequence is as shown in SEQ ID NO:2; Internal primer FIP, its nucleotide sequence is as shown in SEQ ID As shown in NO:3, the inner primer BIP has a nucleotide sequence as shown in SEQ ID NO:4. The concentration of the outer primers F3 and B3 is 0.2 μmol/μl, and the concentration of the inner primers FIP and BIP is 1.2 μmol/μl.

Bst DNA polymerase at a concentration of 8 U/μl;

Nucleic acid dye: 1000×SYBR Green I;

Positive control: ergot genomic DNA;

Negative control: 100 mM Tris-HCl (pH 8.0) and 50 mM EDTA.

Embodiment 2 ergot specific detection

2.1 LAMP-specific detection

2.1.1 Plants to be tested

The tested plants included 8 ergot plants (including 3 rye plants and 5 barley plants), 2 cucumber plants with anthracnose disease, 2 pea plants with powdery mildew and 2 healthy barley plants from Hebei Academy of Agriculture and Forestry Sciences. Ergot formation can be seen on the ears of ergot plants.

2.1.2 Sample pretreatment:

Cut off the ergot plant from the base of the stem, rinse the upper part with sterile water, cut it into small sections with sterile scissors, place it on PDA medium, and cultivate it at a constant temperature of 25 degrees until colonies form.

Use an inoculation loop to scrape off the white mold layer on the leaves of pea plants with powdery mildew, inoculate it on a PDA plate, and culture it by streaking. Use sterilized scissors to cut off an appropriate amount of anthracnose cucumber leaves, and isolate and culture anthracnose bacteria on PDA medium.

Pick a small amount of mycelium of the above-mentioned pathogenic bacteria colony, and observe its morphological characteristics under an optical microscope. Observation showed that the cultured colonies were in line with the colony characteristics of corresponding pathogens.

2.1.3 LAMP detection

Use the kit prepared in Example 1 to detect according to the following steps:

(1) Collect the colonies on the PDA plate and centrifuge, add CTAB extraction buffer, and extract DNA according to the conventional CTAB method;

(2) Prepare a LAMP reaction system in a PCR tube, including 1 μl of each of the four primers, 2.5 μl of reaction buffer, 1 μl of 8 U/μl Bst DNA polymerase, 2 μl of template DNA obtained in step (1), add water to make up to 25 μl, and Ergot Genomic DNA was used as a positive control, and 100mM Tris-HCl pH 8.0 and 50mM EDTA were used as a negative control;

(3) Place the PCR tube in step (2) at a constant temperature of 63°C for 60 minutes;

(4) Analyze and judge the result of the reaction product: add 1 μl of 1000×SYBR GreenI to the reaction product obtained in (3), if the color of the reaction solution is orange, the result is negative, and if the color of the reaction solution is green, the result is positive.

2.2 Test results

The PCR tubes of the 8 ergot diseased plants and the positive control were all green, while the color development results of pea, cucumber, healthy barley and the negative control were all orange, indicating that the primers can accurately identify the diseased plants carrying ergot fungus and have great potential. Strong specificity.

Embodiment 3 ergot sensitivity detection

3.1 LAMP sensitivity test

Extract ergot DNA according to the step (1) of embodiment 2, measure its OD value with ultraviolet spectrophotometer, and be diluted into 10ng, 1ng, 100pg, 10pg, 1pg, 100fg altogether 6 gradients with 10 times concentration serial dilution method.

The LAMP reaction system, reaction conditions and result analysis are the same as steps (2)-(4) in Section 2.1.3 of Example 2.

3.2 Test results

The PCR tubes with ergot DNA concentrations of 10ng, 1ng, 100pg, 10pg, and 1pg all showed green, indicating that the minimum detection limit of the detection method of the present invention reaches 1pg DNA, and the sensitivity is very high.

Claims (1)

1. a kind of LAMP method that detects ergot bacteria, described method is used for identifying the ergot bacteria in food, it may further comprise the steps: (1) Add CTAB extraction buffer to the sample to be tested, and extract DNA according to the conventional CTAB method; (2) Prepare a LAMP reaction system in a PCR tube, which includes 1 μl of outer primer F3 at 0.2 μmol/μl, 1 μl of outer primer B3 at 0.2 μmol/μl, 1 μl of inner primer FIP at 1.2 μmol/μl, and 1 μl of inner primer FIP at 1.2 μmol/μl Primer BIP 1μl, reaction buffer 2.5μl, 8U/μl Bst DNA polymerase 1μl, template DNA 2μl, add water to make up to 25μl, and use ergot genome DNA as positive control, 100mM Tris-HCl pH 8.0 and 50mM EDTA as positive control negative control, Wherein said primer is as follows: Outer primer F3: CGGACACTCAAGATCGACC (SEQ ID NO: 1) Outer primer B3: CGATATCGGGCCTTGTGAAT (SEQ ID NO: 2) Internal primer FIP: TGGGGTTCGTTTCGGCTTGAA-GACAAGCGTGCTTGACCAAT (SEQ ID NO: 3) Internal primer BIP: GAGAATCTGAGGCCGGCTACTG-TCCTTCCTATGCCCTGCT (SEQ ID NO: 4); (3) React the PCR tube in step (2) at a constant temperature of 63°C for 60min; (4) Analyze and judge the result of the reaction product: add 1 μl of nucleic acid dye SYBR Green I to the reaction product obtained in (3), if the color of the reaction solution is orange, the result is negative, and the food does not contain ergot bacteria, if the color of the reaction solution is Green, indicating a positive result.