CN102146477B - Real-time fluorescence PCR (polymerase chain reaction) detection method based on ITS (internal transcribed spacer) gene for Opogona sacchari - Google Patents

Abstract

The invention discloses a real-time fluorescence PCR (polymerase chain reaction) detection method based on ITS (internal transcribed spacer) gene for Opogona sacchari, comprising the following steps of: extracting the DNA of a sample to be detected, preparing the DNA into a template, performing real-time fluorescence PCR by an upstream primer, a downstream primer and a probe, and determining the variety of the sample according to the Ct value. The method is quick, stable and reliable, has high sensitivity, and is suitable for detecting different stages of Opogona sacchari. The method lays a foundation for classification on the molecular level and quick identification of allied species for Opogona (Lepidoptera: Tineidae) in China. The research results can be directly applied to quarantine and identification of Opogona sacchari in plant passed in and out quarantine, thus having great importance for protecting production safety in agriculture and forestry in China and maintaining the ecological balance.

Description

A kind of Opogona sacchari real-time fluorescence PCR detection method based on the ITS gene

Technical field

The present invention relates to a kind of molecular assay method of living species, particularly a kind of Opogona sacchari real-time fluorescence PCR detection method based on the ITS gene.

Background technology

Opogona sacchari Opogona sacchari(Bojer) be the newly-increased quarantine harmful organisms of China, this worm is also classified as quarantine property insect in European Region plant protection tissue (EPPO), Asian-Pacific area plant protection tissue (APPO) and north America region plant protection tissue areas such as (NAPPO).In the ornamental plant of Beijing, found first in 1997.This worm host is extensive, and that has reported has 46 kinds of 24 sections; Can endanger banana, sugarcane, brasiletto Dracaena fragrana(L.), money tree Pachira macrocarpaDeng ornamental plant.It is reported that Opogona sacchari finds that near Madagascar it and northern Seychelles are found this worm in succession then as far back as Mauritius.Soon, this worm is imported South Africa again into.Nineteen twenty-eight, sharp (Canary) archipelago of Ghana on the Atlantic Ocean begins to occur Opogona sacchari, imports Holland in 1972 into, imports Britain and Italy in 1974 into.Found Opogona sacchari in 1975 in South America first, and grow surely in Sao Paulo of Brazil.Late 1980s, Opogona sacchari has the report of distribution in the North America, has distribution in the Florida State and the Hawaiian Islands of the U.S. at present.In addition, Japan and India also find this worm in succession.The right living scope that this worm is described is wide, and colonization ability is strong.

Flat moth genus ( OpogonaZeller, 1853) very similar in shape with its sibling species class, rely on formalness that it is carried out kind merely and identify relatively difficulty.Rely on morphology to identify and require the reviewer need possess the very technology of specialty, and an X-ray inspection X appraiser major part does not possess such technical requirements.In addition, morphology is identified requirement to the worm attitude than higher, and this has also increased its quarantine difficulty virtually.

CN 101787394A discloses through the COI gene in the amplification Opogona sacchari Mitochondrial Genome Overview, and with its importing E.coliMiddle clone purification; Screening afterwards obtains efficiently expressing the gene or the gene fragment of difference between species of characteristic, the Opogona sacchari aggregate species of Opogona sacchari; Design Opogona sacchari Auele Specific Primer CF720 and CR1113 confirm the quarantine result through judging the pulsating situation that exists of specific amplification.This method operation easier is big, be difficult to obtain enough COI genes, the time that follow-up analysis also can labor.

Summary of the invention

The object of the present invention is to provide a kind of Opogona sacchari real-time fluorescence PCR detection method based on the ITS gene.

The technical scheme that the present invention taked is:

A kind of Opogona sacchari real-time fluorescence PCR detection method based on the ITS gene may further comprise the steps:

Get sample to be checked, adopt the SDS method to extract genomic dna, the preparation template DNA ,-20 ℃ of preservations are subsequent use;

Get upper reaches amplimer ITSTF402:5'-CTCCAACCGCCGCCTCCTCCTCAGT-3' and downstream primer ITSTR596:5'-CCAAGTCGCATCCGTGACGGTTCG-3' and probe I TS194TM:5'-FAM-GAACCGCACCCGAGCGCGAGACG-TAMRA-3', Premix Ex Taq and ddH ₂O adds template DNA to be checked, is made into the PCR reaction system, and the TV of reaction system is 25 μ l, and each becomes to be grouped into as follows: 12.5 μ l Premix Ex Taq (2 *), each 0.75 μ l of upstream and downstream primer, concentration is 10pmol/ μ l, the ddH of 5.5 μ l ₂O, the probe of 0.5 μ l, its concentration is 10pmol/ μ l; 5 μ l template DNAs;

The PCR reaction conditions is: 95 ℃ of preparatory sex change of 3min, and 95 ℃ of 10s, 60 ℃ of 30s circulate 45 times;

Read the Ct value and judge detected result.

The inventive method has overcome traditional form and has learned the limitation that detects, and has realized quick, the accurate and stable detection to Opogona sacchari.Simultaneously, detection limit of the present invention is low, can detect the sample of 0.1pg/ μ l concentration, can detect the kind of sample Opogona sacchari more exactly.The inventive method can also be used for the classification of flat moth genus and the Rapid identification of sibling species, is applicable to the detection of different worm attitudes.Help to protect China's agricultural and forestry production safely, keep the eubiosis, guarantee public health security.

Description of drawings

Fig. 1 is the real-time fluorescence PCR detection figure of different samples.

Embodiment

The gene (rDNA) of coding ribosome-RNA(rRNA) is a structure gene the most conservative in the eukaryote; It is arranged to be connected the multiple mode; Each repeats to comprise that (18S encodes respectively in three coding regions; 5.8S with the 28S rna gene); Separated by two ITS (Internal Transcribed Spacers) between the coding region, separated by an ETS (External Transcribed Spacer) and an IGS (Integenic Spacer) respectively between the repetition, owing to rDNA has the difference between hundreds of individual copies (having increased the sensitivity that detects) and ITS and IGS the zone stable and kind in planting in genome; Make it become the important object of many biological heredity Study on Variation; Also be a focus in insect heritable variation in recent years, phyletic evolution and the Molecular Identification research, existing research shows: ITS zone high conservative on the length of base sequence that the same monoid of many insects is not of the same race does not have evident difference; And nucleotide sequence have a height variability, is unusual ideal insect molecular marker gene.

The application has identified Opogona sacchari through the experiment clone O. sacchariTwo geographical population, and 4 sibling species classes O. thiadelpha, O. arista, O. vica, O. nigerovenaKind with 1 relative genus Wegneria cerodeltaThe ITS sequence, hereditary difference is studied between being used for flat moth belonged to kind, it is conservative that the result shows that the ITS sequence belongs to kind of inner height flat moth, and variation is to a certain degree arranged again between kind, is the marker gene that the flat moth of reasonable research belongs to a Phylogenetic Relationships not of the same race.

The quality of primer, reaction system, reaction conditions have direct influence to detected result.

Below in conjunction with embodiment, further specify the present invention.

Get sample to be checked, adopt the SDS method to extract genomic dna, the preparation template DNA ,-20 ℃ of preservations are subsequent use;

Set up the PCR reaction system:

Get specificity amplification primer and specific probe ITS TF402, ITS TR596 and ITS 194 TM; Takara (Dalian is precious biological) Premix Ex Taq (2 *) and ddH ₂O adds template DNA to be checked, is made into the PCR reaction system; The nucleotide sequence of wherein said upstream primer ITS TF402, downstream primer ITS TR596 and primer probe I TS 194 TM is respectively:

Upstream primer ITS TF402:5'-CTCCAACCGCCGCCTCCTCCTCAGT-3'; (SEQ ID NO:1)

Downstream primer ITS TR596:5'-CCAAGTCGCATCCGTGACGGTTCG-3'; (SEQ ID NO:2)

Primer probe I TS 194 TM:5'-FAM-GAACCGCACCCGAGCGCGAGACG-TAMRA-3', wherein FAM is the fluorescence report group, TAMRA is the fluorescent quenching group;

Carry out pcr amplification, the real-time fluorescence PCR reaction conditions is: 95 ℃ of preparatory sex change of 3min; 95 ℃ of 10s then, 60 ℃ of 30s circulate 45 times;

The fluorescent PCR appearance reads the Ct value and judges the result who detects.

The reaction TV of above-mentioned PCR reaction system is 25 μ l, and each becomes to be grouped into as follows: 12.5 μ l Takara premix Ex Taq (2 *); Each 0.75 μ l (10pmol/ μ l) of upstream and downstream primer; 5.5 μ l distilled water; 0.5 μ l probe (10 pmol/ μ l); 5 μ l template DNAs.

Receive this numbering of sample and originate as shown in the table.

The foundation of real-time fluorescence PCR system

TV is 25 μ l, and each becomes to be grouped into as follows: 12.5 μ l Takara premix Ex Taq (2 *); Each 0.75 μ l (10pmol/ μ l) of upstream and downstream primer; 5.5 μ l distilled water; 0.5 μ l probe (10 pmol/ μ l); 5 μ l template DNAs.

The PCR reaction conditions is: 95 ℃ of 3 preparatory sex change of min; 95 ℃ of 10 s then, 60 ℃ of 30 s circulates 45 times.

Wherein, upstream primer is ITS TF402:5'-CTCCAACCGCCGCCTCCTCCTCAGT-3' (SEQ ID NO:1); Downstream primer ITS TR596:5'-CCAAGTCGCATCCGTGACGGTTCG-3' (SEQ ID NO:2); Primer probe I TS 194 TM:5'-FAM-GAACCGCACCCGAGCGCGAGACG-TAMRA-3'.

Supply sample originally to carry out the real-time fluorescence PCR reaction to all, to detect the specificity of ITS TF402/ ITS TR596/ITS 194 TM.The result is as shown in Figure 1, and among the figure, the baseline top is respectively 1-2: O. sacchariThe baseline below is respectively 3: O. thiadelpha4: O. arista5: O. vica6: O. nigerovena7-8: Wegneria cerodelta9:ddH ₂O.

Can be known that by Fig. 1 the amplified fluorescence signal has all appearred in 2 different geographical population samples of Opogona sacchari, the Ct value is greatly about about 18 circulations, but its sibling species O. thiadelpha; O. arista; O. vica; O. nigerovena; Wegneria cerodeltaAll do not have the amplified fluorescence signal in sample, show that this method can carry out specific amplification to Opogona sacchari.

Can know that by above-mentioned experiment present method is a kind of quick, reliable and stable, highly sensitive Opogona sacchari rapid molecular authentication method, be applicable to the detection of the different worm attitudes of Opogona sacchari.This method is that classification and the Rapid identification of sibling species that the flat moth of the lepidopteran rain moth section of China belongs on molecular level laid a good foundation.Result of study can directly apply to the quarantine of Opogona sacchari in the Plant Quarantine of passing in and out and identify, this has very important meaning to protecting China's eagroforestry production safety and keeping eubiosis aspect.

< 110>Zhuhai Entry-Exit Inspection and Quarantine Bureau of P.R.C.

 

< 120>a kind of Opogona sacchari real-time fluorescence PCR detection method based on the ITS gene

 

<130>

 

<160> 3

 

<170> PatentIn?version?3.5

 

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<211> 25

<212> DNA

< 213>artificial sequence

 

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ctccaaccgc?cgcctcctcc?tcagt 25

 

 

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<211> 24

<212> DNA

< 213>artificial sequence

 

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ccaagtcgca?tccgtgacgg?ttcg 24

 

 

<210> 3

<211> 23

<212> DNA

< 213>artificial sequence

<400> 3

gaaccgcacc?cgagcgcgag?acg 23

Claims (1)

1. Opogona sacchari real-time fluorescence PCR detection method based on the ITS gene may further comprise the steps:

Get sample to be checked, adopt the SDS method to extract genomic dna, the preparation template DNA ,-20 ℃ of preservations are subsequent use;

Get upper reaches amplimer ITSTF402:5'-CTCCAACCGCCGCCTCCTCCTCAGT-3' (SEQ ID NO:1) and downstream primer ITSTR596:5'-CCAAGTCGCATCCGTGACGGTTCG-3' (SEQ ID NO:2) and probe I TS194TM:5'-FAM-GAACCGCACCCGAGCGCGAGACG-TAMRA-3', Premix Ex Taq and ddH ₂O adds template DNA to be checked, is made into the PCR reaction system, and the TV of reaction system is 25 μ l, and each becomes to be grouped into as follows: 12.5 μ l Premix Ex Taq (2 *), each 0.75 μ l of upstream and downstream primer, concentration is 10pmol/ μ l, the ddH of 5.5 μ l ₂O, the probe of 0.5 μ l, its concentration is 10pmol/ μ l; 5 μ l template DNAs;

The PCR reaction conditions is: 95 ℃ of preparatory sex change of 3min; 95 ℃ of 10s then, 60 ℃ of 30s circulate 45 times;

Read the Ct value and judge detected result.

Images