CN109825613A - Duck derived components rapid detection method and kit in a kind of food - Google Patents
Duck derived components rapid detection method and kit in a kind of food Download PDFInfo
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- CN109825613A CN109825613A CN201910289655.8A CN201910289655A CN109825613A CN 109825613 A CN109825613 A CN 109825613A CN 201910289655 A CN201910289655 A CN 201910289655A CN 109825613 A CN109825613 A CN 109825613A
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Abstract
The present invention provides duck derived components rapid detection method and kit in a kind of food, is related to biological species identification technology field.The present invention filters out the general internal standard gene LOC106018245 in a duck source first, and nucleotide sequence is as shown in SEQ ID NO.1, and copy number is constant in duck species, and allelic variation is not present, and can be used as the target gene in identification duck 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 duck derived components in food, detection sensitivity is up to 0.2% (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
Technical field
The present invention relates to biological species identification technology fields, more particularly to a kind of ring of duck derived components in detection food
The quick detection kit of mediated isothermal amplification technology (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 duck.Currently, the detection method for food adulteration is mainly
PCR method.PCR method is needed by specific apparatus, and the judgement of final result needs agarose gel electrophoresis to complete, entire mistake
Journey takes a long time.Therefore, it is an object of the present invention to provide a kind of rapid sensitive detection kits of duck 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 quick spirit of duck derived components in food
Quick detection kit.The present invention is without complicated instrument, and the time is short, and easy to operate, high sensitivity can satisfy live inspection completely
The demand of survey.
Summary of the invention
The object of the present invention is to provide the general internal standard genes in duck source for detecting duck 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 duck derived components.
It is a kind of for detecting the gene of duck derived components in food, be internal standard gene LOC106018245, there is SEQ
Sequence shown in ID NO.1.
The present invention provides application of the above-mentioned internal standard gene LOC106018245 in detection duck derived components.
The application that the present invention provides above-mentioned internal standard gene LOC106018245 in food in the identification of duck derived components.
The present invention provides a kind of for detecting the specific LAMP primer group of above-mentioned internal standard gene LOC106018245
It closes, including following 4 primers:
F3:5 '-AGTGTGTGGTAAAAAGGCATG-3 ' (SEQ ID NO.2)；
B3:5 '-TGAAGGAAAGAGCTCCCG-3 ' (SEQ ID NO.3)；
FIP:5 '-GATGGTGAGCGGCTTCCTTAGTCCTGAGGGGTAATTTGCA-3 ' (SEQ ID NO.4)；
BIP:5 '-CAGTGCTTTCCGGCTCAGAGACCTTTAGTCCTTCAGGAACTT-3 ' (SEQ ID NO.5).
5 ' end mark fluorescents of wherein 5 ' end labels biotin (Biotin) of inner primer FIP, BIP are plain (FITC).
The present invention provides application of the above-mentioned specific LAMP primer composition in the identification of duck derived components.
The present invention provides above-mentioned specific LAMP primer compositions in preparation duck derived components detection kit or detection reagent
In application.
Further, the present invention provides a kind of the quick of detection duck derived components containing above-mentioned specific LAMP primer composition
Detection kit.
The present invention provides a kind of method for detecting duck 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.Detection T line and Quality Control C line have 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 terminate reaction.
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 FITC antibody, and nature controlling line is marked with biotin secondary antibody, in conjunction with being lined with colloidal gold-biotin antibody
Marker.
The present invention filters out duck source internal standard gene on chromosome for the first time.The present invention verifies internal standard with multiple kind ducks
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 copy number low
And stablize, general animal internal standard gene often selects on mitochondria, and such reference gene copy number is more, is not easy to quantitative.
For gene on selective staining body of the present invention as internal standard gene, copy number is low, is easy to quantitative, compared to the base on mitochondria
Cause, mutation rate are 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 duck 6 specific regions of specificity internal standard gene target sequence to carry out isothermal duplication.LAMP's
Inner primer FIP and BIP mark biotin (Biotin) and fluorescein (FITC) respectively.It can produce by LAMP reaction a large amount of
Double stranded DNA target substance with biotin and fluorescein.Then LAMP reaction product is detected with colloidal gold nucleic acid test strip.Colloid
Golden nucleic acid test strip p-wire (TL) and nature controlling line (CL) are marked with FITC antibody and biotin secondary antibody respectively, in conjunction with being lined with colloid
Gold-biotin antibody marker.When LAMP reaction product is added drop-wise to test strips sample pad, due to capillarity, product meeting
Successively pass through bonding pad, detection line (TL), nature controlling line (CL), specific binding effect and colloidal gold due to " Ag-Ab "
Coagulation effect, when LAMP reaction product is positive, p-wire (TL) and nature controlling line (CL) have red stripes；Reaction product is yin
When property, 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 colloidal gold core for the mixing of the quality such as duck minced meat and non-duck minced meat progress gradient
Sour test strips are detected to probe into the sensitivity of this detection method.The results show that the detectable limit of detection kit of the present invention is
0.2% (w/w).
Based on present invention determine that for detecting the internal standard gene LOC106018245 of duck derived components in food, the present invention
The LAMP primer composition for detecting the gene is devised, can detect using LAMP reaction bonded colloidal gold nucleic acid test strip to be measured
Whether duck derived components are had in sample, 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
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
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 be duck specificity internal standard gene in 16 kinds of animals LAMP amplification, swimming lane 1 be duck LAMP product, 1:
Duck；2: pig；3: ox；4: sheep: 5: goat；6: chicken；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 duck source LAMP product colloidal gold nucleic acid test strip；1: duck；2:
Chicken；3: ox；4: sheep: 5: pig；6: donkey；7: goose: 8: horse；9: goat；10: fish；11: yak；12: buffalo；13: rat；Sample 1
P-wire (TL) and nature controlling line (CL) there are red stripes then to prove positive sample, sample 2-13 only has nature controlling line (CL) to have
Red stripes are then negative sample；
Fig. 4 is that the detection sensitivity of LAMP- colloidal gold nucleic acid test strip is tested；1: 4 times of gradient of mixing, as original quality
25%；2: 20 times of gradient of mixing, as original quality 5%；3: 100 times of gradient of mixing, as original quality 1%；4: mixing
500 times of gradient, as original quality 0.2%；5: 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 LOC106018245 in 1 duck source of embodiment
By the gene information in search GenBank about duck, target gene group is downloaded from NCBI, and saves as
" .FASTA " format.Analyzed for the full-length genome information of duck, using BLAST and DNAMAN Version4.0 software into
Row homology analysis filters out LOC106018245 gene.By 20 kinds of meats (be common family chicken (Gallus gallus) respectively,
Pheasant (Phasianuscolchicus), turkey (Meleagris 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
Ox (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)) LOC106018245 channel genes DNAMAN Version4.0, carry out multisequencing
Specificity analysis, sequence alignment result save as " .seq " format.The high segment of selection specificity carries out BLAST analysis again, looks into
Look for sequence homology and specificity in database.It is integrated finally by sequence, determines final specific targets gene
LOC106018245 gene can be used as internal standard gene.The nucleotide sequence of the LOC106018245 segment such as SEQ ID
Shown in NO.1.
The foundation of 2 duck 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 V5.0(http://primerexplorer.jp/elamp5.0.0/index.html)
LAMP primer, including 2 outer primers F3, B3 and 2 inner primers are designed for the LOC106018245 gene that embodiment 1 determines
FIP, BIP are shown in Table 1.5 ' end mark fluorescents of 5 ' end labels biotin (Biotin) of inner primer FIP, BIP are plain (FITC).
1 LAMP primer sequence of table
Duck 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, duck
Specific internal standard gene in 16 kinds of animals LAMP amplification, swimming lane 1 be duck LAMP product, 1: duck；2: pig；3: ox；4:
Sheep: 5: goat；6: chicken；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 duck sample, shows LOC106018245 gene and corresponding LAMP
System can be used for the quick detection of duck type.
The foundation of 3 duck 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.
FITC 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 2-13, which only has nature controlling line (CL) to have red stripes,.1: duck；2: chicken；3: ox；4: sheep: 5: pig；6: donkey；7:
Goose: 8: horse；9: goat；10: fish；11: yak；12: buffalo；13: rat；
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 duck minced meat is carried out 5 times with non-duck minced meat (ox, pig, mouse etc. mix minced meat)
The mixing of the quality such as gradient carries out LAMP reaction, then combines with colloidal gold nucleic acid test strip to probe into colloidal gold nucleic acid test paper
The sensitivity of detection method.As a result as shown in figure 4,1: 4 times of gradient of mixing, as the 25% of original quality；2: mixing gradient
20 times, as original quality 5%；3: 100 times of gradient of mixing, as original quality 1%；4: 500 times of gradient of mixing, it is as original
Quality 0.2%；5: negative.It is initial when mixing gradient is 500 times (mixing 500 times that minced meat quality is duck minced meat quality)
Quality 0.2% when, detection T line it is especially shallow, therefore the detection of detection kit of the present invention limit at least can achieve 0.2% (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, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>China Agricultural University
<120>duck derived components rapid detection method and kit in a kind of food
<130> MP1907465Z
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 400
<212> DNA
<213>duck (Anas platyrhynchos)
<400> 1
gggccggtca gtgtgtggta aaaaggcatg gggagggtgt ccgtggtcct gaggggtaat 60
ttgcaccatg gtcaaggcct cactgagcta aggaagccgc tcaccatctc cattcctgct 120
ttgatgacaa aagctgccag tgctttccgg ctcagagcag ctactggctg accataaagt 180
ggccagtgaa gttcctgaag gactaaaggt ggctgaagaa cggggcggga gctctttcct 240
tcagccttag tttgttgtac tagtgggaaa acaatgcttt aacgccataa cctgcagcca 300
gactgccaca ggcacagcct gtcttaggga catgcatgtg gccagaccct gtgtccatcc 360
ctgagccacc aactatcacg gtcccgctgt gtcagcctct 400
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agtgtgtggt aaaaaggcat g 21
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tgaaggaaag agctcccg 18
<210> 4
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gatggtgagc ggcttcctta gtcctgaggg gtaatttgca 40
<210> 5
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cagtgctttc cggctcagag acctttagtc cttcaggaac tt 42
Claims (10)
1. it is a kind of for detecting the gene of duck derived components in food, it is internal standard gene, there is sequence shown in SEQ ID NO.1
Column.
2. application of the gene described in claim 1 in detection duck derived components.
3. application of the gene described in claim 1 in food duck Components identification.
4. the specific LAMP primer composition for detecting gene described in claim 1, including following 4 primers:
F3:5 '-AGTGTGTGGTAAAAAGGCATG-3 ' (SEQ ID NO.2)；
B3:5 '-TGAAGGAAAGAGCTCCCG-3 ' (SEQ ID NO.3)；
FIP:5 '-GATGGTGAGCGGCTTCCTTAGTCCTGAGGGGTAATTTGCA-3 ' (SEQ ID NO.4)；
BIP:5 '-CAGTGCTTTCCGGCTCAGAGACCTTTAGTCCTTCAGGAACTT-3 ' (SEQ ID NO.5).
5. application of the specificity LAMP primer composition as claimed in claim 4 in the identification of duck derived components.
6. specificity LAMP primer composition as claimed in claim 4 is in preparation duck derived components detection kit or detection reagent
Using.
7. the rapid detection method of duck 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；Detection T line and Quality Control C line have 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 FITC antibody, and nature controlling line is marked with biotin secondary antibody, knot
Conjunction is lined with colloidal gold-biotin antibody marker.
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Cited By (1)
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| CN111020013A (en) * | 2019-12-30 | 2020-04-17 | 浙江省农业科学院 | Method for detecting duck-origin nucleic acid component |
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