CN109811066A - Sheep derived components rapid detection method and kit in a kind of food - Google Patents

Abstract

The present invention provides sheep derived components quick detection kit in a kind of food, is related to biological species identification technology field.The present invention filters out the general internal standard gene C alponin in a sheep source first, and nucleotide sequence is as shown in SEQ ID NO.1, and copy number is constant in ovine species, and allelic variation is not present, and can be used as the target gene in identification sheep source.LAMP amplimer is devised by target sequence of the gene, constant-temperature amplification is carried out together with LAMP reaction solution, LAMP reaction product is detected for colloidal gold nucleic acid test strip.The detection of LAMP reaction bonded colloidal gold nucleic acid test strip constitutes quick detection kit, can rapidly and sensitively detect the sheep derived components in food, detection sensitivity is up to 0.16% (w/w).Kit application method of the present invention is simple, low in cost, and reaction result is easy to observe, and specificity is good, is highly suitable for live real-time detection.

Description

Sheep derived components rapid detection method and kit in a kind of food

Technical field

The present invention relates to biological species identification technology fields, more particularly to sheep derived components in a kind of detection food The quick detection kit of loop-mediated isothermal amplification technique (LAMP) association colloid gold nucleic acid test strip detection.

Background technique

With rapid development of economy, the raising of living standards of the people, demand of China resident to meat product increases year by year Add.Although many country's clear stipulaties mark type, the source of meat with requiring food labelling true, unambiguous, forbid adulterated behavior, But still there is the event of many meat adulterations in the market, such as in order to reduce cost, pork, sheep are mixed in donkey fire Beef or pork are mixed in meat, adulterate other meats etc. behavior in sausage.Currently, the detection method for food adulteration is main It is PCR method.PCR method is needed by specific apparatus, and the judgement of final result needs agarose gel electrophoresis to complete, entirely Process takes a long time.Therefore, it is an object of the present invention to provide a kind of rapid sensitive detection kits of sheep derived components in food.

Currently, internal standard gene is widely used for identifying food adulteration, but how to filter out suitable internal standard base Because being particularly important.Current meat products detection of adulterations technology both domestic and external is specifically drawn for the gene design on mitochondria Object carries out real-time fluorescence quantitative PCR amplification, and since chondriogen is multi-copy gene, detection sensitivity is high, but simultaneously There is puzzlement when distinguishing as unconscious cross contamination caused by processes and the conscious illegal addition such as selling, transporting. In addition, the concentration of high copy number mitochondrial DNA can not be corresponding with the concentration of genomic DNA, therefore essence can not be carried out to sample True quantitative analysis, simultaneously because chondriogen homology is high, it is difficult to realize qualitative detection by regular-PCR.Therefore, the party Method can only realize that the screening of meat adulteration identifies by quantitative fluorescent PCR.To identify the cross contamination unintentionally of low concentration and having The illegal addition of meaning and clearly adulterated ratio realization rapid screening, then want the low copy gene on selective staining body as meat internal standard Quasi- gene.

Loop-mediated isothermal amplification (loop-mediated isotherm amplification, LAMP) be by Constant temperature nucleic acid amplification method Notomi novel in one kind of exploitation in 2000, principle are polymerize using a kind of strand displacement DNA Enzyme (BstDNA polymerase) and two pairs of special primers specifically identify 6 isolated areas on target sequence, in isothermal Under the conditions of (65 DEG C or so) heat preservation dozens of minutes, nucleic acid amplification reaction can be completed.

Colloidal gold nucleic acid test strip is the specific binding by antigen and antibody, therefore it is with high sensitivity. The present invention combines LAMP technology with the detection of colloidal gold nucleic acid test strip, provides a kind of the quick of sheep derived components in food Sensitive detection kit.The present invention is without complicated instrument, and the time is short, and easy to operate, high sensitivity can satisfy scene completely The demand of detection.

Summary of the invention

The object of the present invention is to provide the general internal standard genes in sheep source for detecting sheep derived components in food.

Another object of the present invention is to provide a kind of in high sensitivity, high specific, detection food easy to operate The quick detection kit of the LAMP association colloid gold nucleic acid test strip detection of sheep derived components.

It is a kind of for detecting the gene of sheep derived components in food, be internal standard gene C alponin, there is SEQ ID Sequence shown in NO.1.

The present invention provides application of the above-mentioned internal standard gene C alponin in detection sheep derived components.

The application that the present invention provides above-mentioned internal standard gene C alponin in food in the identification of sheep derived components.

The present invention provides a kind of for detecting the specific LAMP primer composition of above-mentioned internal standard gene C alponin, wraps Include following 4 primers:

F3:5 '-CTCACTCCTCTGACAGTCCT-3 ' (SEQ ID NO.2)；

B3:5 '-CCTGGGGCAACAGAATGG-3 ' (SEQ ID NO.3)；

FIP:5 '-GCTAACCTCCAGGCCTCAGTTTGGTGGGTACTGTTACCGTCA-3 ' (SEQ ID NO.4)；

BIP:5 '-AGCTGAACCCTGGAAATCAGGCACTGAGGCTCAGAGAAGGT-3 ' (SEQ ID NO.5).

5 ' end mark fluorescents of wherein 5 ' end labels biotin (Biotin) of inner primer FIP, BIP are plain (Cy5).

The present invention provides application of the above-mentioned specific LAMP primer composition in the identification of sheep derived components.

The present invention provides above-mentioned specific LAMP primer compositions in preparation sheep derived components detection kit or detection examination Application in agent.

Further, the present invention provides a kind of the fast of detection sheep derived components containing above-mentioned specific LAMP primer composition Fast detection kit.

The present invention provides a kind of method for detecting sheep derived components in food, comprising the following steps:

(1) sample to be tested extracts DNA；

(2) to extract DNA as template, LAMP detection is carried out using specificity LAMP primer composition described in claim 3；

(3) result judges: carrying out result judgement using colloidal gold nucleic acid test strip, detecting T line and Quality Control C line has red Band contains target gene；Quality Control C line has red stripes, and detects T line without band, does not contain target gene.

In the above method, step (2) the LAMP detection, the concrete configuration of 25 μ L LAMP detection architectures are as follows: 1 × Thermopol buffer, 0.4mM dNTP, 3mM MgSO₄, 1.0M glycine betaine, 1.6 μM of primers Fs IP, 1.6 μM of primer BIP, 0.2 μM of primers F, 3,0.2 μM of primer B3,8U Bst archaeal dna polymerase large fragment.

In the above method, LAMP detects reaction condition are as follows: then 60-65 DEG C of constant temperature 20min, 85 DEG C of 5min are terminated anti- It answers.

In the above method, colloidal gold nucleic acid test strip described in step (3) include colloidal gold nucleic acid test strip p-wire with Nature controlling line, p-wire are marked with Cy5 antibody, and nature controlling line is marked with biotin secondary antibody, in conjunction with being lined with colloidal gold-biotin antibody mark Remember object.

The present invention filters out sheep source internal standard gene on chromosome for the first time.The present invention is in the verifying of multiple kind sheep Standard gene, it was demonstrated that the internal standard gene is stablized, and allelic variation is not present.The selection of internal standard gene generally requires to copy Number is low and stablizes, and general animal internal standard gene often selects on mitochondria, and such reference gene copy number is more, and it is fixed to be not easy to Amount.For gene on selective staining body of the present invention as internal standard gene, copy number is low, is easy to quantitative, compared on mitochondria Gene, mutation rate is lower.

Fig. 1 is ring mediated isothermal amplification and colloidal gold nucleic acid test strip detection method schematic diagram.The present invention is anti-using LAMP It answers, designs 4 primers for sheep 6 specific regions of specificity internal standard gene target sequence to carry out isothermal duplication.In LAMP Inner primer FIP and BIP mark biotin (Biotin) and fluorescein (Cy5) respectively.It can produce by LAMP reaction a large amount of The double stranded DNA target substance with biotin and fluorescein.Then LAMP reaction product is detected with colloidal gold nucleic acid test strip.Glue Body gold nucleic acid test strip p-wire (TL) and nature controlling line (CL) are marked with Cy5 antibody and biotin secondary antibody respectively, in conjunction with being lined with glue Body gold-biotin antibody marker.When LAMP reaction product is added drop-wise to test strips sample pad, due to capillarity, product Bonding pad, detection line (TL), nature controlling line (CL), specific binding effect and colloid due to " Ag-Ab " can successively be passed through Golden coagulation effect, when LAMP reaction product is positive, p-wire (TL) and nature controlling line (CL) have red stripes；Reaction product is When negative, then only nature controlling line (CL) has red stripes.

Colloidal gold nucleic acid test strip is the specific binding by antigen and antibody, therefore it is with high sensitivity. The present invention is by passing through LAMP reaction bonded for the mixing of the quality such as meat of a sheep minced meat and non-meat of a sheep minced meat 5 times of gradients of progress Colloidal gold nucleic acid test strip is detected to probe into the sensitivity of this detection method.The results show that the detection of detection kit of the present invention The limit is 0.16% (w/w).

Based on present invention determine that for detecting the internal standard gene C alponin of sheep derived components in food, the present invention is set The LAMP primer composition for detecting the gene has been counted, it is detectable to test sample using LAMP reaction bonded colloidal gold nucleic acid test strip Whether sheep derived components are had in product, this rapid reaction is time-consuming few, and specificity is good, and high sensitivity is easy to operate, does not need profession Personnel's operation, is as a result easy to observe, and is very suitable for the use of base's food supervision and inspection.

Detailed description of the invention

Fig. 1 is the principle of the quick detection kit of ring mediated isothermal amplification and the detection of colloidal gold nucleic acid test strip；

Fig. 2 is LAMP amplification of the sheep specificity internal standard gene in 16 kinds of animals, and swimming lane 1 is that the LAMP of sheep is produced Object, 1: sheep；2: ox；3: pig；4: goat: 5: chicken；6: duck；7: goose: 8: horse；9: donkey；10: deer；11: yak；12: buffalo；13: Ermine；14: camel；15: fish；16: rat；M:Maker DL2000；

Fig. 3 is the positive judgement with negative findings in the detection of sheep source LAMP product colloidal gold nucleic acid test strip, sample 1 P-wire (TL) and nature controlling line (CL) have red stripes then to prove positive sample, and sample 2 only has nature controlling line (CL) to have red Band is then negative sample；

Fig. 4 is that the detection sensitivity of LAMP- colloidal gold nucleic acid test strip is tested, 1: 0 times of gradient of mixing, as original quality 100%；2: 5 times of gradient of mixing, as the 20% of original quality；3: 25 times of gradient of mixing, as original quality 4%；4: mixed Close 125 times of gradient, as original quality 0.8%；5: 625 times of gradient of mixing, as original quality 0.16%；6: negative.

Specific embodiment

Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to Range.

Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.

Pig (Sus scrofa), ox (Bos taurus), sheep (Ovis aries), common family chicken (Gallus gallus), Pheasant (Phasianuscolchicus), turkey (Meleagris gallopavo), Gallus domesticlus brisson (Gallus domesticus Brisson), duck (Anas platyrhynchos), goose (Goose calicivirus), dog (Canis lupus Familiaris), rabbit (Oryctolagus cuniculus), yak (Bos mutus), yellow croaker (Pseudosciaena Polyactis it) is bought for supermarket.Horse (Equus caballus), donkey (Equus asinus) are the purchase of Beijing market of farm produce.Always Mouse (Mus musculus) is provided by China Agricultural University's food safety and Molecular Biology Lab.Buffalo (Bubalus Bubalis), ermine (Martes zibellina), camel (Camelus ferus), deer (Cervus) are entered and left the border by Tianjin and are examined Doctor Li Zongmeng of office provides.

The screening of the general internal standard gene C alponin in 1 sheep source of embodiment

By the gene information in search GenBank about sheep, target gene group is downloaded from NCBI, and saves as " .FASTA " format.It is analyzed for the full-length genome information of sheep, utilizes 4.0 software of BLAST and DNAMAN Version Homology analysis is carried out, Calponin gene is filtered out, which is located on chromosome, and coded Ca improves protein gene.By 20 Kind meat (is common family chicken (Gallus gallus), pheasant (Phasianuscolchicus), turkey (Meleagris respectively Gallopavo), Gallus domesticlus brisson (Gallus domesticus brisson), pig (Sus scrofa), ox (Bos taurus), sheep (Ovis aries), duck (Anas platyrhynchos), goose (Goose calicivirus), dog (Canis lupus Familiaris), rabbit (Oryctolagus cuniculus), yak (Bos mutus), yellow croaker (Pseudosciaena Polyactis), horse (Equus caballus), donkey (Equus asinus), mouse (Mus musculus), buffalo (Bubalus bubalis), ermine (Martes zibellina), camel (Camelus ferus), deer (Cervus)) Calponin channel genes DNAMAN Version 4.0, carries out the analysis of multisequencing specificity, and sequence alignment result saves as " .seq " format.The high segment of selection specificity carries out BLAST analysis again, searches sequence homology and specificity in database. It is integrated finally by sequence, determines that final specific targets gene C alponin gene can be used as internal standard base Cause.The nucleotide sequence of the Calponin segment is as shown in SEQ ID NO.1.

The foundation of 2 sheep derived components LAMP detection method of embodiment

Primer Photographing On-line software LAMP primer designing is mediated using Japanese Rong Yan Co., Ltd. ring software primerexplorer V 5.0(http://primerexplorer.jp/elamp5.0.0/index.html) LAMP primer is designed for the Calponin gene that embodiment 1 determines, including 2 outer primers F3, B3 and 2 inner primer FIP, BIP is shown in Table 1.5 ' end mark fluorescents of 5 ' end labels biotin (Biotin) of inner primer FIP, BIP are plain (Cy5).

1 LAMP primer sequence of table

Sheep sample, 25 μ L of reaction system, including 1 × Thermopol buffer, 0.4mM are quickly detected using LAMP DNTP, 3mM MgSO₄, 1.0M glycine betaine, 1.6 μM of primers Fs IP, 1.6 μM of primer BIP, 0.2 μM of primers F, 3,0.2 μM of primer B3, 8U Bst archaeal dna polymerase large fragment.Response procedures are 65 DEG C of constant temperature 1h, 85 DEG C of 5min.After amplification, 2% agarose is utilized Gel electrophoresis carries out product judgement, ladder-like band proof occurs and expands successfully, contains target gene.As a result as shown in Fig. 2, it is continuous Sheep specificity internal standard gene in 16 kinds of animals LAMP amplification, swimming lane 1 be sheep LAMP product, 1: sheep；2: ox；3: Pig；4: goat: 5: chicken；6: duck；7: goose: 8: horse；9: donkey；10: deer；11: yak；12: buffalo；13: ermine；14: camel；15: fish； 16: rat；M:Maker DL2000.Only there is bright band in sheep sample, shows Calponin gene and corresponding LAMP system can be used for the quick detection of sheep type.

The foundation of 3 sheep derived components LAMP product colloidal gold nucleic acid test strip detection method of embodiment

The preparation of colloidal gold labeled monoclonal antibody prepares colloidal gold using trisodium citrate improved method, and is purified with supercentrifugal process Gold labeling antibody stores for future use the gold labeling antibody prepared at 4 DEG C.

Cy5 antibody is diluted to optium concentration with buffer respectively.P-wire (TL) between nature controlling line (CL) at a distance from be 4.5mm is sprayed on NC film respectively by 1.0 μ L/cm.It will be spare after 37 DEG C of NC film sprayed drying overnight.Test strips are cut into 3.8mm wide.

It is added dropwise in the sample pad of colloidal gold nucleic acid test strip after LAMP reaction product and buffer are sufficiently mixed, at this time Mixed liquor passes through bonding pad and NC film under capillary power, and continues, 3min after i.e. observable inspection mobile to water absorption pad direction Survey result.As shown in figure 3, the p-wire (TL) of sample 1 and nature controlling line (CL) have red stripes then to prove positive sample, sample It is then negative sample that product 2, which only have nature controlling line (CL) to have red stripes,.

Colloidal gold nucleic acid test strip is the specific binding by antigen and antibody, therefore it is with high sensitivity. Colloidal gold nucleic acid test strip is closed in advance with the BSA solution that concentration is 3%, before not influencing its normal positive colour developing Put the appearance for avoiding false positive.By the way that meat of a sheep minced meat is carried out with non-meat of a sheep minced meat (ox, pig, mouse etc. mix minced meat) The mixing of the quality such as 5 times of gradients carries out LAMP reaction, then combines with colloidal gold nucleic acid test strip to probe into the examination of colloidal gold nucleic acid The sensitivity of paper slip detection method.As a result as shown in figure 4,1: 0 times of gradient of mixing, as the 100% of original quality；2: mixing ladder 5 times of degree, as the 20% of original quality；3: 25 times of gradient of mixing, as original quality 4%；4: 125 times of gradient of mixing, as Original quality 0.8%；5: 625 times of gradient of mixing, as original quality 0.16%；6: negative.When mixing gradient is 625 times (mixed Closing minced meat quality is 625 times of meat of a sheep minced meat quality) when being the 0.16% of initial mass, detection T line is especially shallow, Ji Huyu Negative control is similar, therefore the detection of detection kit of the present invention is limited to 0.16% (w/w).

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Sequence table

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tatgcttagc attttcggta ccttaactca ctcctctgac agtcctaggg ggtgggtact 180

gttaccgtca tgccctttat acagatggga aaactgaggc ctggaggtta gcacacttgc 240

taggattgct caggtgatga gtagctgaac cctggaaatc aggccactta gcagtcatgt 300

gacacacctt ctctgagcct cagtttccat tctgttgccc caggggggtg gaaacagggc 360

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cctggggcaa cagaatgg 18

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Claims (10)

1. it is a kind of for detecting the gene of sheep derived components in food, it is internal standard gene, has shown in SEQ ID NO.1 Sequence.

2. application of the gene described in claim 1 in detection sheep derived components.

3. application of the gene described in claim 1 in food sheep Components identification.

4. the specific LAMP primer composition for detecting gene described in claim 1, including following 4 primers:

F3:5 '-CTCACTCCTCTGACAGTCCT-3 ' (SEQ ID NO.2)；

B3:5 '-CCTGGGGCAACAGAATGG-3 ' (SEQ ID NO.3)；

FIP:5 '-GCTAACCTCCAGGCCTCAGTTTGGTGGGTACTGTTACCGTCA-3 ' (SEQ ID NO.4)；

BIP:5 '-AGCTGAACCCTGGAAATCAGGCACTGAGGCTCAGAGAAGGT-3 ' (SEQ ID NO.5).

5. application of the specificity LAMP primer composition as claimed in claim 4 in the identification of sheep derived components.

6. specificity LAMP primer composition as claimed in claim 4 is in preparation sheep derived components detection kit or detection reagent Application.

7. the rapid detection method of sheep derived components in a kind of detection food, which comprises the following steps:

(1) sample to be tested extracts DNA；

(2) to extract DNA as template, LAMP detection is carried out using specificity LAMP primer composition described in claim 4；

(3) result judges: carrying out result judgement using colloidal gold nucleic acid test strip, detecting T line and Quality Control C line has red bar Band contains target gene；Quality Control C line has red stripes, and detects T line without band, does not contain target gene.

8. the method for claim 7, which is characterized in that step (2) the LAMP detection, 25 μ L LAMP detect body The concrete configuration of system are as follows: 1 × Thermopol buffer, 0.4mM dNTP, 3mM MgSO₄, 1.0M glycine betaine, 1.6 μM of primers FIP, 1.6 μM of primer BIP, 0.2 μM of primers F, 3,0.2 μM of primer B3,8U Bst archaeal dna polymerase large fragment.

9. method as claimed in claim 7 or 8, which is characterized in that LAMP detects reaction condition are as follows: 60-65 DEG C of constant temperature Then 20min, 85 DEG C of 5min terminate reaction.

10. method as claimed in claim 7 or 8, which is characterized in that colloidal gold nucleic acid test strip described in step (3) includes Colloidal gold nucleic acid test strip p-wire and nature controlling line, p-wire are marked with Cy5 antibody, and nature controlling line is marked with biotin secondary antibody, knot Conjunction is lined with colloidal gold-biotin antibody marker.