CN101376911A - Primer sequence for identifying Aspergillus parasiticus and Aspergillus flavus and identification method thereof - Google Patents

Abstract

The invention discloses a primer sequence used to identify Aspergillus parasiticus and Aspergillus flavus; the primer sequence comprises two sets of primers; the forward primer in the first set of primers is provided with the base sequence of SEQ ID NO:1 in a sequence table, and the reverse primer of the first set of primers is provided with the base sequence of SEQ ID NO:2 in the sequence table; the forward primer in the second set of primers is provided with the base sequence of SEQ ID NO:3 in the sequence table, and the reverse primer of the second set of primers is provided with the base sequence of SEQ ID NO:4 in the sequence table. The primer sequence has high specificity, identities Aspergillus parasiticus and Aspergillus flavus through PCR amplification with high speed and reliability, thereby laying theoretical foundation for researching the soil polluted by aflatoxin or the distribution and dynamic changes of two types of fungi in crops and providing scientific bases for the control of aflatoxin.

Description

Be used to identify primer sequence and the authentication method thereof of Aspergillus parasiticus and flavus

Technical field

The present invention relates to biology field, relate in particular to the primer sequence and the authentication method thereof that are used to identify Aspergillus parasiticus and flavus.

Background technology

Aspergillus fungi Aspergillus parasiticus (Aspergillus parasiticus) is close with flavus (A.flavus) phenotypic characteristic and all can produce serious carcinogenic meta-bolites aflatoxin.Aflatoxin is the strongest class biotoxin of finding so far of pollution agricultural-food toxicity.Wherein, what toxicity was the strongest, harm is maximum is aflatoxin B1, and its toxicity is 10 times of potassium cyanide, 68 times of arsenic, is put into the special highly toxic substance of strict control.The agricultural-food aflatoxin contamination has become influences food safety and the healthy stealthy killer of harm people.

At present very difficult to the evaluation of Aspergillus parasiticus and flavus, main adopt traditional morphological method, identified differentiation according to the thickness of the arrangement mode of conidiophore, conidial shape, conidium wall and smooth degree and the color and the form of bacterium colony on a series of substratum (czapek's solution (CZ), flavus differential medium (AFPA) and coconut palm are starched substratum (CCA)).

Existing big quantity research has been reported the detection method of toxin producing aspergillus flavus strain, but never reports the molecular assay method of superparasitism aspergillus and flavus.Time-consuming, the effort of traditional authentication method.

Summary of the invention

The invention provides the primer sequence that is used to identify Aspergillus parasiticus and flavus, this primer specificity height can Rapid identification Aspergillus parasiticus and flavus.

Be used to identify the primer sequence of Aspergillus parasiticus and flavus, comprise two groups of primers, the forward primer of first group of primer has the described base sequence of SEQ ID NO:1 in the sequence table, and the reverse primer of first group of primer has the described base sequence of SEQ ID NO:2 in the sequence table; The forward primer of second group of primer has the described base sequence of SEQ ID NO:3 in the sequence table, and the reverse primer of second group of primer has the described base sequence of SEQ ID NO:4 in the sequence table.

The total sugar of Aspergillus parasiticus and flavus utilizes the glcA gene order in the gene cluster, and other fungi this gene order not, first group of primer is exactly partial sequence in the glcA gene order, utilize first group of primer that the genomic dna of Aspergillus parasiticus and flavus is carried out pcr amplification, can obtain the dna segment of 745-bp, the DNA of other fungi be carried out pcr amplification then can not obtain any dna segment.

Aspergillus parasiticus can produce B1, B2, G1 and four kinds of toxin of G2, flavus can only produce B1 and two kinds of toxin of B2, the toxin synthetic gene that produces malicious Aspergillus parasiticus and produce malicious flavus bunch is discovered, toxin synthetic gene norB, the cypA or their intervening sequences between the two that produce in the malicious flavus lack, and produce malicious Aspergillus parasiticus and then contain complete toxin synthetic gene bunch.

As shown in Figure 1, produce malicious flavus and between norB and cypA, have two kinds of disappearance types (I and II):

Disappearance type i: the 1-280 amino acids, the 1-112 amino acids of cypA gene and the transcribed spacer of norB and cypA that produce malicious flavus disappearance norB.

Disappearance Type II: the 1-181 position and 300-310 amino acids, the 1-29 amino acids of cypA gene and the transcribed spacer (as shown in Figure 1) of norB and cypA that produce malicious flavus disappearance norB gene.

Second group of primer is exactly two kinds of partial sequences that the disappearance type is produced the total deletion fragment of malicious flavus, utilize second group of primer that the Aspergillus parasiticus genomic dna is carried out pcr amplification, can obtain the dna fragmentation of 462-bp, the DNA of other fungi or flavus be carried out pcr amplification then can not obtain any dna segment.

The present invention also provides a kind of method of utilizing above-mentioned primer sequence to identify Aspergillus parasiticus and flavus, may further comprise the steps:

(1) DNA of extraction bacterium to be measured;

(2) DNA with bacterium to be measured is a template, uses first group of primer and second group of primer to carry out pcr amplification;

(3) develop the color with EB behind the PCR product gel electrophoresis,, judge the type of bacterium to be measured according to the DNA band quantity on the gel;

When DNA band quantity is 2, judge that bacterium to be measured is an Aspergillus parasiticus;

When DNA band quantity is 1, judge that bacterium to be measured is a flavus;

When DNA band quantity is 0, judge that bacterium to be measured is other fungi or bacterium.

The invention provides the primer sequence that is used to identify Aspergillus parasiticus and flavus, this sequence-specific height, identify Aspergillus parasiticus and flavus by utilizing this primer sequence by pcr amplification, speed is fast, the reliability height, be distribution and the dynamic change based theoretical of two kinds of fungies in research soil of aflatoxin contamination or the farm crop, and then provide scientific basis for the improvement of aflatoxin.

Description of drawings

Fig. 1 is the position collection of illustrative plates of primer sequence of the present invention on the norA-cypB gene order;

Fig. 2 is the gel electrophoresis spectrum of each bacterial strain pcr amplification product in the specific detection.

Embodiment

Selected bacterial strain

Alternaria brassica (Alternaria brassicae), wild cabbage chain lattice spores (A.brassicicola), Fusarium oxysporum (Fusarium oxysporum), Fusarium graminearum (F.graminearum), rhizoctonia cerealis (Rhizotonia cerealis) Botrytis cinerea (Botrytis cinerea), penicillium digitatum (Penicillium digitatum), drupe brown rot germ (Monilinia fructicola) and 3 Aspergillus parasiticus bacterial strain (SD1-1, SX6-6 and ZJHZ27) and 5 aspergillus flavus strain (SD3-6, SD5-1, SX6-8, GS10-1 and ZJHZ2).

Above-mentioned bacterial strains is preserved in biotechnology research institute of Zhejiang University, also can obtain by the plate streaking separation and purification of routine, and above-mentioned bacterial strains also can be selected other bacterial strain certainly just as experiment material, to not influence of the invention process.

Extract DNA

Scrape from the PDA flat board with inoculating needle and to get mycelia (100mg), place 1.5-mL Eppendorf pipe, add 500 μ L DNA extraction lysate (200mM Tris-HCl, 50mM EDTA, 20mMNaCl, 1%SDS, pH8.0), fully grind vibration mixing, the static 10min of room temperature with electric drill; 13200r/min4 ℃, centrifugal 5min; Get the about 400 μ L of supernatant liquor in new 1.5-mL Eppendorf pipe, add 750 μ L dehydrated alcohols, mixed mixing, 13200r/min4 ℃, centrifugal 5min abandons supernatant; Precipitation is used 70% washing with alcohol, and room temperature is placed dry 5-10min, is dissolved in 30 μ LTE (pH8.0), and-20 ℃ of preservations are standby.

The DNA of above-mentioned 8 kinds of fungies and Aspergillus parasiticus and flavus all adopts aforesaid method to extract.

Primer is synthetic

Utilize glcA gene order in the gene cluster according to the total sugar of Aspergillus parasiticus and flavus, designed primer sets GLF1 and GLR1; Described in summary of the invention, genomic dna with respect to Aspergillus parasiticus, toxin synthetic gene norB, cypA and intervening sequence between the two in the genomic dna of flavus lack, the disappearance type is divided into two kinds, common deletion fragment according to these two kinds disappearance types has designed primer sets NCF2 and NCR2.

The sequence corresponding relation is as shown in the table in the concrete sequence of above-mentioned primer and the sequence table:

The primer title	Sequence	Sequence number in the sequence table
			GLF1	5’-AAG?ACA?CAG?TCA?TCG?CCT?GTT-3’	SEQ?ID?NO.1
GLR1	5’-ACG?CCT?TTA?TCG?AGC?CAA?TA-3’	SEQ?ID?NO.2
			NCF2	5’-ATT?CGC?CAA?ACA?TCT?CTC?CGT-3’	SEQ?ID?NO.3
NCR2	5’-CAC?AGT?CAA?GGC?CAC?CAA?CAA-3’	SEQ?ID?NO.4

Physical location is as shown in Figure 1 on the norA-cypB gene order for above-mentioned primer.The primer of above-mentioned sequence is synthetic by the Shanghai lottery industry, also can be synthetic by conventional primer synthetic method.

Identification of strains

DNA with above-mentioned all bacterial strains is a template, carries out the PCR reaction with GLF1/GLR1 and two groups of primers of NCF2/NCR2, and each reaction all comprises a negative control (replacing dna profiling with sterilized water).

25 μ L reaction systems: 1 μ LDNA template (about 0.4ng), each 0.2 μ mol1 of primer ^-1, dNTP0.2 μ mol1 ^-1, MgCl ₂2mmol1 ^-1, 1 * damping fluid (east, Beijing victory company produces), a polysaccharase 1.5U unit, distilled water complement to 25 μ L.

Reaction conditions is: 95 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations, and last 72 ℃ are extended 5min.The PCR product with 1.5% agarose in 1 * TAE damping fluid behind the electrophoresis, with EB (ethidium bromide)) colour developing takes pictures.

From the electrophoresis photo (as shown in Figure 2), the amplified production of Aspergillus parasiticus develops the color after gel electrophoresis, manifests two DNA bands on the gel, i.e. 745-bp and 462-bp amplified production; The amplified production of flavus develops the color after gel electrophoresis, only manifests a DNA band, i.e. 745-bp amplified production, and the amplified production of other fungi develops the color after gel electrophoresis, does not manifest the DNA band on the gel, i.e. explanation any product that do not increase.

SEQUENCE?LISTING

＜110〉Zhejiang University

＜120〉be used to identify primer sequence and the authentication method thereof of Aspergillus parasiticus and flavus

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<160>4

<170>PatentIn?version?3.3

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<212>DNA

＜213〉Aspergillus parasiticus (Aspergillus parasiticus)

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<212>DNA

＜213〉Aspergillus parasiticus (Aspergillus parasiticus)

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<210>3

<211>21

<212>DNA

＜213〉Aspergillus parasiticus (Aspergillus parasiticus)

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<211>21

<212>DNA

＜213〉Aspergillus parasiticus (Aspergillus parasiticus)

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Claims (2)

1, is used to identify the primer sequence of Aspergillus parasiticus and flavus, comprise two groups of primers, the forward primer of first group of primer has the described base sequence of SEQ ID NO:1 in the sequence table, and the reverse primer of first group of primer has the described base sequence of SEQ ID NO:2 in the sequence table; The forward primer of second group of primer has the described base sequence of SEQ ID NO:3 in the sequence table, and the reverse primer of second group of primer has the described base sequence of SEQ ID NO:4 in the sequence table.

2, a kind of method of identifying Aspergillus parasiticus and flavus may further comprise the steps:

(1) DNA of extraction bacterium to be measured;

(2) DNA with bacterium to be measured is a template, uses first group of primer and second group of primer to carry out pcr amplification;

(3) develop the color with EB behind the PCR product gel electrophoresis,, judge the type of bacterium to be measured according to the DNA band quantity on the gel;

When DNA band quantity is 2, judge that bacterium to be measured is an Aspergillus parasiticus;

When DNA band quantity is 1, judge that bacterium to be measured is a flavus;

When DNA band quantity is 0, judge that bacterium to be measured is other fungi or bacterium.

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