AU2021103536A4 - Primer probe set for Taqman fluorescent PCR assay and application - Google Patents
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The present invention discloses a primer-probe set for Taqman fluorescent PCR assay and
its application, which relates to the field of biotechnology; the invention designs specific
primers and MGB fluorescent probes separately, optimizes the reaction system and conditions,
and the combination of three primer-probe systems can simultaneously detect triple fluorescent
PCR methods for fish, mammalian and avian-derived components, with the lowest detection
limit as low as 0.02ng genome/reaction, with intra- and inter-group coefficients of variation
less than 2%. The Taq man triple RT-qPCR method established by the present invention has
the advantages of specificity, high sensitivity and good reproducibility, which provides a new
technical analysis means for the qualitative analysis of animal products including feeds in terms
of their source components.
Description
Primer probe set for Taqman fluorescent PCR assay and application
The present invention relates to the field of biotechnology, and in particular to a
primer probe set for Taqman fluorescent PCR assay and applications.
Animal products raw materials and processed products, especially animal-derived
food and agricultural products, as well as animal-derived feed products, substandard,
adulterated and unintentional pollution and other phenomena are common, such as fish
meal adulterated with lower-priced chicken meal or feather meal. In order to ensure the
authenticity of the relevant animal-derived products, domestic and foreign researchers
have established a series of testing methods for animal-derived ingredients such as
cattle, sheep, pigs, chickens, ducks, geese and fish, and also some standards specify the
technical methods for the detection and identification of fish, mammalian and avian
derived ingredients. Research and identification methods for the analysis of animal
derived components have gradually increased, such as sensory discrimination,
microscopic methods, colorimetric methods, chemical testing methods, spectroscopic
techniques, etc.. However, since all methods have certain limitations, they cannot better
meet the detection needs. At this time, nucleic acid detection methods target the animal
nucleic acid genome, and the technology is constantly updated and iterated, the
advantages of nucleic acid detection methods can be highlighted.
In summary, despite the emergence of new animal-derived components detection
technology, however, there is not yet a simple and easy to use multiple testing and identification methods, can be used to quickly analyze feed and other animal products in fish, mammals, Avian-derived ingredients, accurate determination of fish meal, meat and bone meal adulteration to make false phenomenon.
The purpose of the present invention is to provide a primer probe set for Taqman
fluorescent PCR assay and application to solve the problems of the prior art described
above.
To achieve the above, the present invention provides a primer probe set for
Taqman fluorescent PCR assay, said primer probe set comprising at least one of the
followings:
(1) Fish primer probe set: Fish-F: GAATGGCTCATTAAATCAGTTATGGT;
Fish-R:CGGCATGTATTAGCTCTAGAATTACC;Fish-P:FAM
CCTTTGATCGCTC-BHQ1;
(2) Mammalian primer probe set: Mal-F: CCGATAACGAACGAGACTCTG;
Mal-R: GGCTGAACGCCACTTGTC; Mal-P: Hex-TCCCCCAACTTC-BHQ2;
(3) Avian primer probe set: Avi-F: GCATTCGTATTGYGCCGC; Avi-R:
TACGACGGTATCTGATCGTCT; Avi-P: Cy5-CAAGACGAACTAAAG-BHQ2.
The present invention also provides an application of the described primer probe
set for Taqman fluorescent PCR assay in the preparation of Taqman fluorescent PCR
assay products.
The present invention also provides a Taqman fluorescent PCR assay kit, the
Taqman fluorescent PCR assay kit comprising said primer probe set for Taqman
fluorescent PCR assay.
The present invention also provides a Taqman fluorescent PCR detection method,
the Taqman fluorescent PCR detection method is: Taqman fluorescent PCR detection
of the sample using the primer probe set for Taqman fluorescent PCR detection.
Further, the Taqman fluorescent PCR reaction system for the Taqman fluorescent
PCR assay method is any of the following volume ratios:
(1) Single-weight fluorescent PCR reaction system: 2 X Fluorescent PCR Buffer:
pmol/L primer probe set: nucleic acid template: RNase-free Water = 10:2.5:2:5.5.
(2) Triple fluorescent PCR reaction system: 2 X Fluorescent PCR Buffer: 10
pmol/L Mammalian primer probe set: 10 pmol/L Fish primer probe set: 10 pmol/L
Avian primer probe set: Nucleic acid template: RNase-free Water = 10: 1.4: 1.4: 1.4:
1.4: 3: 2.8.
Further, the volume ratio of upstream primer, downstream primer and probe in the
primer probe group in said (1) is 1:1:0.5; The volume ratio of upstream primer,
downstream primer and probe in the primer probe group in the mammalian primer
probe group, fish primer probe group or avian primer probe group in said (3) is 3:3:1.
The present invention discloses the following technical effects:
The present invention designs specific primers and MGB fluorescent probes
respectively, and optimizes the reaction system and conditions so that each primer
probe system can specifically detect the target species type without cross-reacting with other species, e.g. fish probes can specifically detect cod, perch and carp materials without cross-reacting with pig, cow, sheep, chicken and goose materials. The combination of the three primer-probe systems allows the simultaneous detection of fish, mammalian and avian-derived components by triple fluorescent PCR with a minimum detection limit as low as 0.02 ng genome/reaction and intra- and inter-group coefficients of variation less than 2%.
Using the triple fluorescence PCR method established in this study, 35 imported
and domestic feed samples were tested and two domestic beef bone meal were found
to have detected components of avian origin. The results show that the Taq man triple
RT-qPCR method established in this study has the advantages of specificity, high
sensitivity and good reproducibility, and provides a new technical analysis tool for the
qualitative analysis of animal products including feeds in terms of their source
components.
Figure 1 shows the results of triple fluorescence PCR sensitivity test; where, A is
fish; B is mammal; C is avian.
Figure 2 shows the triple fluorescence PCR standard curve; where, A is fish; B is
mammal; and C is avian.
If not otherwise specified, the materials in the embodiments of the invention are
commercially available.
Example 1 DNA extraction
For raw meat samples, the surface layer was removed with sterile, wipe-clean
surgical scissors (to avoid surface contamination), chopped and used for nucleic acid
DNA extraction.
Accurately weigh 20mg of meat and feed samples and extract them according to
the instructions of the DNA Genome Extraction Kit DNeasy Blood & Tissue Kit.
Example 2 Design of Primers and Probes
Avian 18S rRNA sequences in Genbank were collected (Anas platyrhynchos
AF173614.1, Gallus gallus DQ018752.1, Meleagris gallopavo AJ419877.1, Struthio
camelus AF173607.1), Mammalian 18S rRNA sequences (Bos taurus NR036642.1,
Sus scrofaNR046261.1, Ovis ariesKY129860.1, Euus caballusAJ311673.1), Fish 18S
rRNA sequences ( Solea senegalensis EF126042.1, Merluccius merluccius
KF986699.1, Trachurus trachurus KF986703.1, Scomber scombrus KF986714.1,Liza
subviridis KJ774647.1, Oncorhynchus tshawytscha KJ774722.1,Oreochromis niloticus
XR003218722.1, Gadus morhua XR003975744.1 ), Shrimps (Panulirus homarus
GQ411530.1, Panulirus argus AY743955.1, Penaeus semisulcatus DQ079766.1,
Macrobrachium lanchesteri KP725756.1, Palaemon serratus KP725812.1,
Procambarus clarkia KX444578.1, Euphausia mutica DQ900739.1), shellfish (Pinctada
imbricata AB214464.1, Pinctada fucata AB214463.1, Chlamys islandica L11232.1,
Mytilus trossulus L24490.1, and Ostrea edulis L49052.1), etc.
Design principle, take avian universal probe as an example: apply MEGA7.0
clustalW program to compare the above sequences and select target sequences, which
are consistent with 18S rRNA sequences of duck, chicken and turkey, and different from other mammals, fish, shrimp and shellfish; After selecting the probe, avian universal amplification primers were selected upstream and downstream using
Taqman-MGB probe primer design principles.
The probes designed for the study were all MGB probes, with a length of 12 to 18
bases, and such probes can distinguish between 1 and 2 bases of difference. The
collected fish, mammalian, avian, shrimp and shellfish 18SrRNA sequences were
homology matched, and the probes were manually designed based on the selection of
the corresponding regions, including four sets of fish probe primers, six sets of
mammalian primer probes and four sets of avian primer probes. The extracted nucleic
acid samples were applied for specificity detection, and the best specific primer probe
systems were screened for one set each for mammalian, fish and avian species (Table
1)
Table 1 Triple fluorescent PCR primer probe design and sequence
Primers and Probes Primer/probe sequences (5'-3')
Mal-F CCGATAACGAACGAGACTCTG
Mal-R GGCTGAACGCCACTTGTC Mal-P Hex-TCCCCCAACTTC-BHQ2(MGB)
Fish-F GAATGGCTCATTAAATCAGTTATGGT
Fish-R CGGCATGTATTAGCTCTAGAATTACC
Fish-P FAM-CCTTTGATCGCTC-BHQ1(MGB)
Avi-F GCATTCGTATTGYGCCGC
Avi-R TACGACGGTATCTGATCGTCT
Avi-P
Cy5-CAAGACGAACTAAAG
BHQ2(MGB)
Example 3 Real-time fluorescence PCR reaction (application of QIAGEN
Quantinova Probe PCR Mix (fluorescent type) kit)
In this study, we first established three sets of single-weight fluorescent PCR
assays for three types of animals: fish, mammals and avian, meeting the following
requirements: no mutual interference between primers/probes, amplification and
excitation of fluorescent signals only for target sequences of specific species of animals,
and no amplification and fluorescent signals for non-target sequences. The triple
fluorescence PCR reaction conditions were finalized by optimizing the triple
fluorescence PCR reaction temperature, reaction time, primer probe concentration and
other conditions.
Single-weight fluorescent PCR reaction system (20 pL): 2 XFluorescent PCR
Buffer 10 L, 10 pmol/L upstream and downstream primers 1 L each, 10 pmol/L
fluorescent probe 0.5 pL, nucleic acid template 2 pL, RNase-free Water 5.5 pL.
Triple fluorescent PCR reaction system (20 pL) for detection of single and double
samples: 2 Fluorescent PCR Buffer 10 pL, 10 gmol/L upstream and downstream
primers (Mal-F, Mal-R, Fish-F, Fish-R, Avi-F, Avi-R) 0.6 pL each, 10 tmol/L probes
(Mal-P, Fish-P, Avi-P) 0.2 pL each, nucleic acid template 2 pL, RNase-free Water 3.8
pL.
Triple fluorescent PCR reaction system (20 tL) for detecting triple samples: 2 X
Fluorescent PCR Buffer 10 pL, 10 tmol/L upstream and downstream primers (Mal-F,
Mal-R, Fish-F, Fish-R, Avi-F, Avi-R) 0.6 L each, 10 pmol/L probes (Mal-P, Fish-P,
Avi-P) 0.2 L each, 3 L of nucleic acid template, and 2.8 L of RNase-free Water.
The reaction conditions were set to 95 °C for 2 min; 95 °C for 5 s and 60 °C for
s, for a total of 35 cycles. Fluorescence signals were collected at the annealing stage.
Example 4 Specificity Experiment
Single-weight samples: Single nucleic acid samples extracted from lamb, beef,
pork, whole chicken, duck breast, turkey leg, whole goose, salmon, snapper, tilapia,
golden pomfret sea bass, cod, carp and grass carp were tested by applying the triple
fluorescent PCR system established in this study at a concentration of 10 ng/4L and a
final concentration of 20 ng/reaction. rNase-free Water was used as a negative control.
Dual samples: The triple fluorescent PCR system established in this study was
applied to detect dual mixed nucleic acid samples: goose/sheep, chicken/bovine,
duck/pig, chicken/pig, goose/salmon, goose/cod, chicken/tilapia, duck/grass carp,
duck/perch, sheep/salmon, bovine/cod, pig/grass carp, sheep/seabream, bovine/grass
carp, pig/pumpkin, and the concentration of each nucleic acid sample was 10 ng/4, 1
pL of each nucleic acid sample was added to the PCR system. RNase-free Water was
used as a negative control.
Triple samples: The triple fluorescent PCR system established in this study was
applied to detect triple mixed nucleic acid samples: duck/sheep/salmon,
chicken/pig/seabass, turkey/bull/tilapia, and chicken/sheep/seabream. The
concentration of each nucleic acid sample was 10 ng/4L, and 1 L of each nucleic acid
sample was added to the PCR system. RNase-free Water was used as a negative control.
The results are shown in Table 2. Only the corresponding positive controls showed S
type typical amplification curves with Ct values less than 35, while no amplification
curves were observed for the negative controls or other species categories, indicating
that the triple fluorescence PCR method established in this study has good specificity.
Table 2 Results of triple fluorescent PCR system for single, double and triple
samples
Sample Sample Triple fluorescent PCR assay results
combinations Fish Mammal Avian
Single-weight goose - -
+ samples turkey - -
+ chicken - -
+ duck - -
+ sheep - +
pig - + cow - + Salmon + -
Snapper + -
Oreochromis niloticus + -
Golden pomfret + -
gadus + -
Carp + -
Sea Bass + -
Grass carp + -
Keyhole shrimp - -
White shrimp - -
Oyster - -
White clam - - -
Dual samples Goose+Sheep - +
+ Turkey+Cow - +
+ Duck+Pig - +
+ Chicken+Pig - +
+ Goose+Salon + -
+ Goose+Gadus + -
+ Chicken+reochromis niloticus + -
+ Duck+Grass carp + -
+ Duck+Sea Bass + -
+ Sheep+Salon + + Cow+Gadus + + Pig+Grass carp + + Sheep+Snapper + +
Cow+Grass carp + + Pig+Golden pomfret + + Triple samples Pig+Goose+Gadus + +
Chicken+Cow+Grass carp + + + + Duck+Sheep+Salon + + +
Chicken+Pig+Sea bass + + +
Turkey+Cow+Oreochromis niloticus + + +
Chicken+Sheep+Snapper + + +
Where "+" means a positive test result and "-" means a negative test result.
Example 5 Sensitivity test and the establishment of standard curve
The extracted sample nucleic acid was adjusted to1OOng/piL, and then the genomic
nucleic acid was diluted with RNase-free Water in a10-fold concentration gradient with
final concentrations of lOng/tL, Ing/tL, 0.lng/tL, 0.0lng/tL, and 0.001ng/pL,
respectively. Using RNase-free Water as a negative control, the sensitivity of the triple fluorescence PCR method was examined by applying the above-mentioned multiplicative dilutions of nucleic acid templates and selecting the most common bovine, cod, and chicken-derived components in feeds as templates using the triple fluorescence PCR system established in this study. The results are shown in Figure 1.
The minimum detection limit of this fluorescent PCR system was 0.02 ng/reaction for
bovine genomic samples, 0.002 ng/reaction for cod genomic samples, and 0.02
ng/reaction for chicken genomic samples. The test results showed that the minimum
genomic detection limits were different for different species, e.g. for carp and cod
(results not shown).
Based on the above sensitivity tests, the standard curves were plotted and the
results are shown in Figure 2: the correlation coefficients R2 of the standard curves for
fish (cod), mammals (cattle) and avian (chicken) were 0.9998, 0.9997 and 0.9996,
respectively, with good linearity. The regression equations of the standard curves for
fish, mammals and avian are y=-3.3024x+24.525, y=-3.5652x+28.629 and y=
3.4776x+29.025 respectively. Therefore, the samples can be determined as negative or
positive based on the Ct values of the samples examined and with reference to the
standard curves.
Example 6 Repeatability test
Selected mixed chicken/pig/cod samples were used as templates at 10 ng/4L, 1
ng/4L, and 0.1 ng/L, and each concentration was replicated six times, using the triple
fluorescence PCR method established in this study; The coefficient of variation was
calculated from the Ct values obtained from the test results to assess the reproducibility of the method. The results are shown in Table 3, and the coefficient of variation of the repeated tests for chicken/pig/cod was less than 2%, indicating that the triple fluorescence PCR method established in this study was reproducible.
Table 3 Intra-group and inter-group reproducibility results of the triple fluorescence
PCR method
Sample Sample concentration/( Repeatability test ng/reactioon) Average( Coefficient of M+SD) variation (CV) /% Fish( 20 20.22±0.14 0.71 Cod) 2 23.34±0.27 1.17 0.2 27.01±0.36 1.35 Mammal 20 24.17±0.22 0.91 (Pig) 2 27.45±0.30 1.09 0.2 31.29±0.42 1.34 avian( 20 24.5332±0.18 0.72 Chicken 2 28.1493±0.24 0.85
0.2 31.3705±0.45 1.43
Example 7 Feed sample testing and compliance testing with testing methods in
industry standards
The nucleic acid samples were extracted from the feed and the concentration was
adjusted to 1Ong/tL, and the triple fluorescent PCR method established in this study
was applied to detect animal-derived components. A mixed chicken/pig/cod sample
(1Ong/4L) was used as a positive control and RNase-free Water was used as a negative
control. Meanwhile, the above feed nucleic acid samples were tested by applying the
standard described method and analyzed for compliance with the triple fluorescence
PCR method to verify the usefulness of the triple fluorescence PCR method established
in this study. The standards involved are "PCR method for the qualitative detection of
fish-derived ingredients in feed" (SN/T 2867-2011), "Real-time fluorescence PCR
method for the qualitative detection of mammalian-derived ingredients in animal
derived feed" (GB/T 21103-2007), "Real-time fluorescence PCR method for the
detection of avian-derived ingredients in feed" (SN/T 2727-2010). 2010).
The test results are shown in Table 4. The results of the composition analysis of
33 of the feed samples were consistent with their original labeled ingredients, while the
remaining three samples were suspected to be adulterated with ingredients of other
species, namely: Mammalian (Ct value 23.67+0.35) and fish-derived components (Ct
value 33.68+0.42) were detected in Uruguayan beef bone meal 3# sample. New Zealand
fish meal 16 # samples detected fish (Ct value 21.45 0.23) and avian-derived
ingredients (Ct value 33.89 0.39); domestic pig blood meal 1 # samples detected
mammals (Ct value 26.37 0.32) and avian-derived ingredients (Ct value 24.56±
0.28). The above test results were verified to be consistent with the test results of the
test method described in the industry standard, with a 100% compliance rate.
Table 4 Results of triple fluorescence PCR assay for feed samples Feed Region of Triple fluorescent PCR samples origin assay results Fish Mammal avian
Port offers Fish Meal 1 Russia + -
FishMeal2 America + - Fish Meal 3 Peru + - Fish Meal 4 Peru + - Fish Meal 5 Peru + - Fish Meal 6 Peru + - Fish Meal 7 Peru + - Fish Meal 8 Peru + - Fish Meal 9 Fish Meal10 Peru + -
Fish Meal 11 Peru + -
Fish Meal 12 Chile + -
Fish Meal 13 Chile + -
Fish Meal 14 Chile + -
Fish Meal 15 India + -
Fish Meal 16 New Zealand + -
Fish Meal 17 New Zealand + - +* Fish Meal 18 Mexico + - Beef bone meal I Denmark + -
Beef bone meal 2 Uruguay - + Beef bone meal 3 Uruguay - + Beef bone meal 4 Uruguay +* + Chicken meal 1 Argentina - + Chicken meal 2 America - - +
Chicken meal 3 America - - +
Chicken meal 4 America - - +
America - - +
Domestic Pig Blood meal I Domestic - + +
Purchase Chicken bone meal 1 Domestic - + +
Beef bone meal5 Domestic - + +
Beef bone meal6 Domestic - + +
Beef bone meal7 Domestic + - +
Fish meal19 Domestic + - +
Fish meal20 Domestic + - +
Fish meal 21 Domestic + - +
Fish meal 22 Peru + -
+ Fish meal 23 Peru + +
Where "+" represents a positive test result, "-" represents a negative test result,
"+*"represents a weakly positive test result, and Ct value > 33.
The above described embodiments are only a description of the preferred way of
the present invention, not a limitation of the scope of the present invention. Without
departing from the spirit of the design of the present invention, all kinds of deformations
and improvements made to the technical solutions of the present invention by a person
of ordinary skill in the art shall fall within the scope of protection determined by the
claims of the present invention.
Claims (6)
1. A primer probe set for a Taqman fluorescent PCR assay, characterized in that
said primer probe set comprises at least one of the followings:
(1) Fish primer probe set:
Fish-F: GAATGGCTCATTAAATCAGTTATGGT;
Fish-R: CGGCATGTATTAGCTCTAGAATTACC;
Fish-P: FAM-CCTTTGATCGCTC-BHQ1;
(2) Mammalian primer probe set:
Mal-F: CCGATAACGAACGAGACTCTG;
Mal-R: GGCTGAACGCCACTTGTC;
Mal-P: Hex-TCCCCCAACTTC-BHQ2;
(3) Avian primer probe set:
Avi-F: GCATTCGTATTGYGCCGC;
Avi-R: TACGACGGTATCTGATCGTCT;
Avi-P: Cy5-CAAGACGAACTAAAG-BHQ2.
2. A primer probe set for Taqman fluorescent PCR assay of claim 1 in the
preparation of a Taqman fluorescent PCR assay product.
3. A Taqman fluorescent PCR assay kit, characterized in that Taqman fluorescent
PCR assay kit comprises the primer probe set for Taqman fluorescent PCR assay as
described in claim 1.
4. A Taqman fluorescent PCR detection method, characterized in that Taqman
fluorescent PCR detection method is: Taqman fluorescent PCR detection of a sample using a primer probe set for Taqman fluorescent PCR detection as described in claim
1.
5. The Taqman fluorescent PCR assay method according to claim 4, characterized
in that the Taqman fluorescent PCR reaction system of said Taqman fluorescent PCR
assay method is any one of the following volume ratios:
(1) Single-weight fluorescent PCR reaction system: 2 X Fluorescent PCR Buffer:
pmol/L primer probe set: nucleic acid template: RNase-free Water = 10:2.5:2:5.5;
(2) Triple fluorescent PCR reaction system: 2 X Fluorescent PCR Buffer: 10
pmol/L Mammalian primer probe set: 10 pmol/L Fish primer probe set: 10 pmol/L
Avian primer probe set: Nucleic acid template: RNase-free Water = 10: 1.4: 1.4: 1.4:
1.4: 3: 2.8.
6. The Taqman fluorescent PCR assay according to claim 5, characterized in that
the volume ratio of upstream primer, downstream primer and probe in the primer probe
group in said (1) is 1:1:0.5; the volume ratio of upstream primer, downstream primer
and probe in the primer probe group in the mammalian primer probe group, fish primer
probe group or avian primer probe group in said (3) is 3:3:1.
FIGURES 1/2
Figure 1
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