AU2021101224A4 - A LAMP detection method for identifying mink in beef and mutton - Google Patents
A LAMP detection method for identifying mink in beef and mutton Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
Abstract
The invention relates to a LAMP detection method for identifying mink meat in beef and
mutton, and belongs to the technical field of molecular biological detection. According to the
detection method, DNA of meat to be detected is used as the template, and DAF3, DAB3,
DAFIP and DABIP are used as primers to construct a LAMP detection system; In the LAMP
detection system, LAMP reaction is carried out in a constant temperature water bath at 64°C
for 40min, and 1 L of fluorescent dye SYBR Green I is added to the reaction solution after the
reaction. If the reaction solution turns green, it shows that there is mink meat in the meat to be
tested; If it is orange, there is no mink meat in the meat to be tested. Compared with the
traditional PCR, the detection method of the invention can be completed in a common constant
temperature water bath for 40 minutes, and the results can be directly observed with naked eyes
by adding dyes into the final products, so that animal-derived components can be quickly and
accurately identified, and the method has the advantages of simple operation, low requirements
on experimental instruments and easy result judgment.
Description
A LAMP detection method for identifying mink in beef and mutton
The invention relates to a LAMP detection method for identifying mink meat in beef
and mutton, and belongs to the technical field of molecular biological detection.
In recent years, with the increasing demand for beef and mutton in the market and the
rapid price increase, some illegal traders, driven by their interests, adulterated other cheap
meat to pretend to be beef and mutton. Since 2012, there have been many incidents in China
in which pork, chicken and duck were used as beef and mutton. In some fur-based special
economic animal breeding areas, the whereabouts of mink meat and fox meat became a
mystery, and many people speculated that they were mixed into beef and mutton and
entered people's dining tables. This phenomenon not only disturbs the economic order, but
also seriously endangers people's health.
At present, the methods used to detect animal-derived components mainly include
routine PCR, fluorescence quantitative PCR, etc., but the above diagnostic methods take a
long time, and the judgment results need precise instruments and equipment, so it is
difficult to popularize and apply them in actual production.
In order to solve the above technical problems existing in detecting mink meat mixed
with beef and mutton by conventional PCR and fluorescence quantitative PCR, the mink
meat is identified by LAMP technology and cytochrome C oxidase subunit I (COX I) gene on animal mitochondrial DNA, and the whole reaction can be carried out in a constant temperature water bath for 40min, and the results can be directly observed with naked eyes by adding dye to the final product.
Technical scheme:
A LAMP detection method for identifying mink meat in beef and mutton by using
mitochondrial DNA, which takes DNA of meat to be detected as a template and DAF3,
DAB3, DAFIP and DABIP as primers to construct a LAMP detection system; The LAMP
detection system reacts in a constant temperature water bath at 64°C for 40min, and after
the reaction, 1 L of fluorescent dye SYBR Green I is added into the reaction solution; If
the reaction solution turns green, it shows that there is mink meat in the meat to be tested;
If it is orange, there is no mink meat in the meat to be tested.
The primers used are as follows:
DAF3: GCTATGGGCCTTAGGGTT, as shown in SEQ ID NO.1.
DAB3: CATTTAGGGTGTAGCCTGT, as shown in SEQ ID NO.2.
as shown in SEQ ID NO.3.
DABIP:GCACATTTTCACTACGTTCTTTCAAGGAACCAGTGAACGAATCC,a
s shown in SEQ ID NO.4.
The LAMP detection system has a total volume of25pL; Its composition is as follows.
0.5pL of DAF3 and DAB3 at 10pmol / L, 4pL of DAFIP and DABIP at 10lmol / L,
4pL of dNTP at 2.5mmol/L, 2pL of MgSO4 at 50mmol / L, 2.5pL of Betaine at 5mol / L, l pL of Bst DNA polymerase, and 2.5 pL of 10 x Thermo buffer, and the rest was ultrapure water.
Compared with the traditional PCR, the detection method of the invention can be
completed in the ordinary constant temperature water bath for 40 min, and can directly
observe the results with naked eyes by adding dye into the final product, which can quickly
and accurately identify the animal derived components; it has the advantages of simple
operation, low requirements for experimental instruments, and easy determination of
results. The invention does not need the help of large expensive instruments and equipment,
which is more suitable for the local detection of grass-roots units, and has broad market
prospects and greater social and economic benefits.
According to the invention, cytochrome C oxidase subunit I (Cox I) gene on
mitochondrial DNA is selected as a target gene; With DAF3, DAB3, DAFIP and DABIP
as primers, COX I gene of mink meat can be specifically amplified, and then mink meat
can be detected from mutton and beef. It can be distinguished from the meat of similar
species such as cats and foxes, and has the advantage of high specificity. In addition, using
DAF3, DAB3, DAFIP and DABIP as primers, the sensitivity is high, and the lowest
detectable concentration is 2x10-4ng/pL. Therefore, even if it is doped with trace amounts
of mink meat, it can be detected.
The invention also provide a detection reagent used by that LAMP detection method,
which comprise the following primers:
DAF3: GCTATGGGCCTTAGGGTT, as shown in SEQ ID NO.1.
DAB3: CATTTAGGGTGTAGCCTGT, as shown in SEQ ID NO.2.
as shown in SEQ ID NO.3.
as shown in SEQ ID NO.4.
The LAMP detection kit for the identification of mink in beef and mutton by using
mitochondrial DNA contains 0.5ipL of DAF3 and DAB3 at 10pmol / L, 4pL of DAFIP and
DABIP at 10pmol / L, 4pL of dNTP at 2.5mmol/L, 2pL of MgSO4 at 50mmol / L, 2.5pL
of Betaine at 5mol / L, l pL of Bst DNA polymerase, and 2.5pL of 10 x Thermo buffer.
DAF3: GCTATGGGCCTTAGGGTT, as shown in SEQ ID NO.1.
DAB3: CATTTAGGGTGTAGCCTGT, as shown in SEQ ID NO.2.
as shown in SEQ ID NO.3.
as shown in SEQ ID NO.4.
Fig. 1 is the graph of detection results of embodiment 1; From left to right: negative
control, positive control, beef, mutton, dog meat, cat meat, fox meat and mink meat; The
results show that the second and eighth tubes are green and the rest are orange;
Fig. 2 is the schematic diagram of sensitivity of PCR detection method; From right to
left: Marker, 10-1, 10-2, 1-,- 1 0 -4, 10- dilutions;
Fig. 3 is the schematic diagram of sensitivity of LAMP detection method; The order
from left to right is: Marker, 10-1, 10-2, 10- , 10-4 and 10-5 dilutions.
In the following examples, all reagents used are analytically pure except those directly
provided by the manufacturer; Water, unless otherwise specified, is ultrapure water.
Example 1 Primers design
The COX I sequence of mink was retrieved from GenBank, and the software was
compared and analyzed to determine the accuracy of the sequence; primers were designed
using PrimerExplore software based on the above sequence as follows:
DAF3: GCTATGGGCCTTAGGGTT (As shown in SEQ ID NO.1).
DAB3: CATTTAGGGTGTAGCCTGT (As shown in SEQ ID NO.2).
(As shown in SEQ ID NO.3).
(As shown in SEQ ID NO.4).
Example 2 Specificity experiment
(1) Extracting sample DNA as the template
The fresh muscle tissues of cattle, sheep, dogs, cats, foxes and minks were collected
strictly according to the sterile method as samples, and the DNA in the samples was
extracted by phenol chloroform extraction method. The LAMP reaction system was established by ultrapure water ( negative control ), positive plasmid ( positive control) containing mink COX I gene preserved in our laboratory, beef DNA, mutton DNA, dog
DNA, cat DNA, fox DNA, and mink DNA as templates; The concentration of DNA
template was 20ng / pL.
( 2 ) Establishment of LAMP reaction system
The LAMP reaction system was established by exploring the concentration ratio of
internal and external primers, dNTP concentration, reaction temperature and reaction time:
L. The composition was as follows: 0.5pL of DAF3 and DAB3 at 10pmol / L, 4pL of
DAFIP and DABIP at 10pmol / L, 4pL of dNTP at 2.5mmol/L, 2pLof MgSO4 at 50mmol
/ L, 2.5 pL of Betaine at 5mol / L, 1 pL of Bst DNA polymerase, and 2.5 pL of 10 x Thermo
buffer, 2pL of Template with minimum concentration of 20ng/ptL, the rest was ultrapure
water.
Among them, dNTP and MgSO4 were purchased from TaKaRa (Dalian ) Co., Ltd.,
Betaine was purchased from Sigma, USA, Bst DNA polymerase ( 8 U /pL ), 10 x Thermo
buffer was purchased from NEB, USA, and SYBR Green I nucleic acid dye was purchased
from Solarbio, Beijing.
LAMP amplification reaction
The above LAMP reaction system ( according to the sequence of template obtained
in Case 1 ) was simultaneously placed in a constant temperature water bath at 64 °C for
LAMP amplification for 40 min. 1 pL fluorescent dye SYBR Green I was added after the
reaction; The color of reaction solution is orange, green, orange, orange, orange, orange, orange, orange, orange, orange, green ( as shown in Figure 1 ). Among them, orange was negative and green was positive.
Example 3
30 fresh beef samples and 30 fresh mutton samples were collected from
slaughterhouses in Shandong Province; 10 mink meat samples were collected from
Shandong Zoological Hospital. The DNA of samples was extracted by the method of
example 1 as template, LAMP reaction system was established and LAMP amplification
was carried out. The detection results were as follows:
Table 1
Beef Mutton Mink Beef and Beef and Mutton meat mutton minkmeat and mink meat
Number 30 30 10 30 10 10 of samples
Positive 0 0 10 0 10 10 number
Positive 0 0 100 0 100 100 rate%
It can be concluded from Example2 and Example 3 that the primer of Example 1 can
specifically amplify mink DNA. Only when the template contains mink DNA, the reaction
solution after adding fluorescent dye SYBR GREEN I to the LAMP reaction product shows
green. The template contains any other non-mink meat ( cattle, sheep, cat, foxe, dog).
Example 4 Sensitivity experiment
The DNA used in the positive control of case 1 was diluted with 101, 10-2, 10- , 10-4
and 10-' dilutions, respectively. The diluted DNA was used as a template for LAMP and
PCR, respectively. The sensitivity of the two detection methods was compared. The results
showed that the amplification sensitivity of LAMP method was higher than that of ordinary
PCR method. The third dilution gradient could be detected by ordinary PCR ( Fig. 2);
LAMP method can still be detected in the fourth dilution gradient ( Figure 3 ).
<110> Animal Products Quality and Safety Center of Shandong Province
< 120 > LAMP detection method for identifying mink meat in beef and mutton
<160>4
<210>1
<211>18
<212>DNA
< 213 > Artificial synthesis
<400>1
GCTATGGGCC TTAGGGTT 18
<210>2
<211>19
<212>DNA
< 213 > Artificial Sequence
<400>2
CATTTAGGGT GTAGCCTGT 19
<210>3
<211>45
<212>DNA
<213>Artificial Sequence
<400>3
GTGAAGAACG ATGTCTAGTG ATGAGTTTTA TTTACAGTGG GTGGC 45
<210>4
<211>44
<212>DNA
<213>Artificial Sequence
<400>4
GCACATTTTC ACTACGTTCT TTCAAGGAAC CAGTGAACGA ATCC 44
Claims (3)
1. A LAMP detection method for identifying mink meat in beef and mutton by using
mitochondrial DNA is characterized in that a LAMP detection system is constructed by
taking DNA of meat to be detected as a template and DAF3, DAB3, DAFIP and DABIP
as primers; The LAMP detection system reacts in a constant temperature water bath at 64°C
for 40min, and after the reaction, 1 L of fluorescent dye SYBR Green I is added into the
reaction solution; If the reaction solution turns green, it shows that there is mink meat in
the meat to be tested; If it is orange, there is no mink meat in the meat to be tested.
The primers used are as follows:
DAF3: GCTATGGGCCTTAGGGTT, as shown in SEQ ID No.1.
DAB3: CATTTAGGGTGTAGCCTGT, as shown in SEQ ID NO.2.
DAFIP:GTGAAGAACGATGTCTAGTGATGAGTTTTATTTACAGTGGGTGGC,
as shown in SEQ ID NO.3.
DABIP:GCACATTTTCACTACGTTCTTTCAAGGAACCAGTGAACGAATCC,
as shown in SEQ ID NO.4.
The lamp detection system has a total volume of 25pL and the composition is as
follows.
0.5pL of DAF3 and DAB3 at 10pmol / L, 4pL of DAFIP and DABIP at 10lmol / L,
4pL of dNTP at 2.5mmol/L, 2pL of MgSO4 at 50mmol / L, 2.5pL of Betaine at 5mol / L,
l pL of Bst DNA polymerase, 2.5pL of 10 x Thermo buffer, 2pL of template and the rest
was ultrapure water
2. The detection reagent used in LAMP detection method for identifying mink meat
in beef and mutton by using mitochondrial DNA is characterized by comprising the
following primers:
DABIP:GCACATTTTCACTACGTTCTTTCAAGGAACCAGTGAACGAATCC,
shown in SEQ ID NO.4.
3. The LAMP detection kit for the identification of mink in beef and mutton by using
mitochondrial DNA is characterized in that it contains 0.5pL of DAF3 and DAB3 at
ptmol / L, 4pL of DAFIP and DABIP at 10pmol / L, 4pL of dNTP at 2.5mmol/l, 2pL of
MgSO4 at 50mmol / L, 2.5pL of Betaine at 5mol / L,1 pL of Bst DNA polymerase, and
2.5pL of 10 x Thermo buffer.
DAFIP:GTGAAGAACGATGTCTAGTGATGAGTTTTATTTACAGTGGGTGGC,
as shown in SEQ ID NO.3.
FIGURES
1/2 2021101224
Figure 1
Figure 2
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| Publication Number | Publication Date |
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| AU2021101224A4 true AU2021101224A4 (en) | 2021-05-06 |
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