Primer group for rapidly and visually identifying Atlantic salmon and rainbow trout by combining nanogold with loop-mediated isothermal amplification, detection method and application

Abstract

The invention relates to the technical field of biology, in particular to a primer group for rapidly and visually identifying Atlantic salmon and rainbow trout by combining nanogold with loop-mediated isothermal amplification, a detection method and application. The primer group comprises an outer primer pair F3 and B3, an inner primer pair FIP and BIP, a loop primer pair LF and LB and a nanogold probe, the sequences of the outer primer pair F3 and the outer primer pair B3 are as shown in SEQ ID No. 1 and SEQ ID No. 2; the sequences of the inner primer pair FIP and the inner primer pair BIP are as shown in SEQ ID No.3 and SEQ ID No.4; the sequences of the loop primer pair LF and the loop primer pair LB are as shown in SEQ ID No. 5 and SEQ ID No. 6; and the DNA (deoxyribonucleic acid) sequence of the nanogold probe is shown as SEQ ID No.7. The method can simply, quickly and visually solve the problem that the Atlantic salmon and rainbow trout species are difficult to accurately and quickly distinguish in the prior art, and is more economical and practical.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a primer set, a detection method and an application for rapid visual identification of Atlantic salmon and rainbow trout by gold nanoparticle combined loop-mediated isothermal amplification. Background Art

[0002] Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are similar in appearance and taste. Especially after being processed into fish fillets or sashimi, their colors, textures and fat distributions are relatively close, making it difficult for consumers to distinguish them by sensory perception alone. Compared with rainbow trout, Atlantic salmon has a higher price. On the one hand, since Atlantic salmon is farmed in seawater, there is no risk of parasite infection when eaten raw, while rainbow trout farmed in fresh water may cause parasite infection when eaten raw. In addition, the heavy metal content in rainbow trout farmed in fresh water is relatively high. Passing off rainbow trout as Atlantic salmon may involve food safety issues.

[0003] Traditional species identification methods often require professional knowledge and techniques, which are difficult for ordinary consumers to master. Although molecular biology techniques such as DNA barcoding can provide accurate identification results, they are not popular in daily consumption.

[0004] Patent application CN2019113731559 discloses "A method for identifying salmon sashimi and rainbow trout sashimi farmed in fresh water". Although this technical solution can detect the contents of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) in fish meat to show the quality of the fish meat, the credibility is insufficient due to only these two characteristic indicators. Compared with the molecular detection method, the accuracy in identifying salmon fillets and rainbow trout fillets is not high. Patent application CN202010093317X discloses "A primer set, a kit and a detection method for detecting Atlantic salmon". However, dyes such as SYBR Green I used therein often affect the amplification reaction, and the cost of fluorescence quantitative PCR detection is relatively high. More importantly, the specificity based on dye detection is greatly limited: since non-specific amplification often occurs during the amplification process, and dye molecules cannot specifically recognize the sequences of specific amplification, it often leads to inaccurate results.

[0005] Therefore, there is an urgent need to establish a more rapid and accurate detection means for convenient species identification of Atlantic salmon and rainbow trout. Summary of the Invention

[0006] The present invention provides a primer set, a detection method and an application for rapid visual identification of Atlantic salmon and rainbow trout by nano-gold combined loop-mediated isothermal amplification. Based on the binding and visual detection characteristics of nucleic acid molecules on the surface of nano-gold and specific amplification sequences, this method can simply and visually solve the problem that it is difficult to quickly and accurately distinguish Atlantic salmon and rainbow trout species in the prior art, solve the problem of Atlantic salmon adulteration, safeguard food safety and the interests of consumers, and is more economical and practical in operation.

[0007] One of the technical solutions adopted by the present invention is:

[0008] A primer set for rapid visual identification of Atlantic salmon and rainbow trout by nano-gold combined loop-mediated isothermal amplification is provided, including an outer primer pair F3 and B3, an inner primer pair FIP and BIP, a loop primer pair LF and LB, and a nano-gold probe;

[0009] The sequences of the outer primer pair F3 and B3 are shown as SEQ ID No.1 and SEQ ID No.2;

[0010] The sequences of the inner primer pair FIP and BIP are shown as SEQ ID No.3 and SEQ ID No.4;

[0011] The sequences of the loop primer pair LF and LB are shown as SEQ ID No.5 and SEQ ID No.6;

[0012] The DNA sequence of the nano-gold probe is:

[0013] 5’-CTAGCAGGTAATCTTGCCCACAAAAAA-3’, as shown in SEQ ID No.7.

[0014] Furthermore, the nano-gold probe is prepared by mixing oligoA-tailed DNA and nano-gold in a molar ratio of not less than 400:1; the size of the nano-gold is 10 - 50nm.

[0015] Furthermore, the nano-gold probe is prepared by mixing oligoA-tailed DNA and nano-gold in a molar ratio of 600:1; the size of the nano-gold is 20nm.

[0016] Another technical solution adopted by the present invention is:

[0017] A kit for rapid visual identification of Atlantic salmon and rainbow trout by nano-gold combined loop-mediated isothermal amplification is provided, including the primer set as described above.

[0018] Furthermore, the above-mentioned kit also includes Buffer, dNTP, MgSO4, and Bst DNA polymerase.

[0019] The third technical solution adopted by the present invention is as follows:

[0020] A detection method for rapid visual identification of Atlantic salmon and rainbow trout by combining nano-gold with loop-mediated isothermal amplification is provided, including the following operation steps:

[0021] (1) Construct an amplification system

[0022] The amplification system includes the primer set as described above, as well as Buffer, dNTP, MgSO4, Bst DNA polymerase, water and template;

[0023] (2) Place the above amplification system in a constant temperature water bath at 65 °C and react for 60 min. After taking it out, add a certain volume of 5 μM MgCl2 solution. After 5 min, identify the detection result according to the color change: when the color always remains red, it indicates that the identified species or meat product is Atlantic salmon; if the color changes from red to blue, it indicates that the identified species or meat product is rainbow trout.

[0024] The fourth technical solution adopted by the present invention is as follows:

[0025] Provide the application of the primer set and kit as described above in the identification of Atlantic salmon and rainbow trout.

[0026] Furthermore, when the above application is used for identification, the color development time is 5 min to distinguish between Atlantic salmon and rainbow trout.

[0027] Advantages of the present invention:

[0028] 1. Aiming at the problem of frequent false positives in the current identification methods for Atlantic salmon and rainbow trout, the present invention combines a specific probe with LAMP amplification to achieve the purpose of simple, visual and highly accurate identification of the two kinds of fish, and can combat the damage to consumers' rights and interests caused by fish adulteration.

[0029] 2. The present invention is simple to operate, low in cost, low in equipment dependence, and high in sensitivity. The constructed constant temperature visual detection system for Atlantic salmon has high sensitivity and strong convenience. Specifically, by designing a primer probe set with special sequences for this detection and performing high-density modification on the probe sequences, the specificity is strong. In the identification of mixed samples or single samples of Atlantic salmon and rainbow trout, the two products can be sensitively, quickly and visually distinguished, avoiding the influence of false positives during primer amplification on the detection results. Description of the drawings

[0030] Figure 1 It is the experimental principle diagram of the present invention;

[0031] Figure 2 It is the comparison of the CO1 genes of Atlantic salmon and rainbow trout of the present invention;

[0032] Figure 3 Schematic diagram of the amplification effect of the first set of primers for Atlantic salmon screened for the present invention;

[0033] Figure 4 Schematic diagram of the amplification effect of the second set of primers for Atlantic salmon screened in the experiment of the present invention;

[0034] Figure 5 Schematic diagram of the color development effect of identifying Atlantic salmon and rainbow trout using the present invention. Detailed implementation manners

[0035] To clearly illustrate the technical features of the present solution, the present invention will be elaborated in detail below through specific implementation manners in conjunction with the accompanying drawings. In the embodiments of the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The experimental methods, detection methods, etc. involved in the following embodiments are all conventional experimental methods and detection methods existing in the prior art without special instructions.

[0036] By comparing the CO1 genes of Atlantic salmon and rainbow trout on BLAST, it is found that their similarity is 87%( Figure 2 ). Taking the CO1 gene unique to Atlantic salmon as the target sequence (Table 1 below shows the sequence information), since the length of the amplified sequence cannot be too long, in this experiment, it was finally determined to amplify a partial fragment of the C01 gene with a length of 237 bp (the specific sequence is the underlined part in Table 1).

[0037] Table 1

[0038]

[0039]

[0040] Then, primer design was carried out on the website https: / / lamp.neb.com / #! / . The gene of Atlantic salmon was input into the website, and the parameters were adjusted to leave sites for probes, resulting in four groups of primers with Atlantic salmon being positive. Primer comparison was carried out on SnapGene, and finally two of them were selected for the experiment.

[0041] I. Primer design

[0042] Design principle: Since the target sequence region is rich in GC content, the GC content of the primers is controlled at 50 - 60%, the Tm value of the primers is controlled at 60°C - 65°C, and the optimal primer size and the distance between the primer binding regions are selected; make the Tm values of F1c and B1c slightly higher than those of F2 and B2 to facilitate the immediate formation of a circular structure when the single-stranded template is released; to improve the primer stability, in addition to avoiding the secondary structures that appear, make the content of dG in the 6 bases at the 5' end of F1c (or B1c) and the 3' ends of F2, F3 (or B2, B3) less than -4 Kcal / mol.

[0043] The information of two sets of primers screened using the above design principles is as follows:

[0044] The sequence information of the first set of primers:

[0045] F3: 5'-CTTCTCCCTCCCTCCTTTC-3' (SEQ ID No.1)

[0046] B3: 5'-TGGGGTTTGATACTGAGAGA-3' (SEQ ID No.2)

[0047] FIP: 5'-AGGGGGGTAGACTGTTCATCCTTCTCCTCCTGGCCTCAT-3'

[0048] (SEQ ID No.3)

[0049] BIP: 5'-GCAGGAGCTTCCGTTGACTTTGGCCCCAAGAATTGAAGA-3' (SEQ ID No.4)

[0050] LF: 5'-AGCGCCGGCTTCAACTC-3' (SEQ ID No.5)

[0051] LB: 5'-CTATTTTTTCCCTCCATTTGGCTGG-3' (SEQ ID No.6)

[0052] The sequence information of the second set of primers:

[0053] F3: 5'-CTGAGCCGGAATAGTCGG-3' (SEQ ID No.8)

[0054] B3: 5'-GGGGGAATGCTATGTCGG-3' (SEQ ID No.9)

[0055] FIP: 5'-TGGTCATCTCCCAGAAGGGCCACCGCCCTAAGTCTCTT-3'

[0056] (SEQ ID No.10)

[0057] BIP: 5'-CAGCCCATGCCTTCGTCATAATCAGTTTCCAAAGCCGC-3'

[0058] (SEQ ID No.11)

[0059] LF: 5'-CTGGCTGAGTTCTGCTCGAAT-3'(SEQ ID No.12)

[0060] LB: empty

[0061] Table 2 below shows the LAMP amplification system. The amplification effects of two sets of primers were screened using ABI7500. By observing the amplification fluorescence curves, it was found that there were significant differences between the two sets of primers. The set of primers with the best amplification effect was selected for the subsequent color reaction.

[0062] Table 2

[0063]

[0064] See Figure 3 , which is a schematic diagram of the amplification effect of the first set of primers. It can be seen from this figure that the peak starts earlier when using this set of primers. When the template concentration is 50 ng / μL, the peak start time of the positive group (template DNA is Atlantic salmon) is much earlier than that of the negative group (template DNA is rainbow trout) and the blank control group, and the specificity is better.

[0065] The amplification effect of the second set of primers is as shown in Figure 4 . It can be seen from the figure that when the template concentration is 50 ng / μL, the peak start time of the positive group (template DNA is Atlantic salmon) is only slightly earlier than that of the negative group (template DNA is rainbow trout) and the blank control group, and the false positives are very serious.

[0066] II. Visual detection by combining LAMP with AuNPs

[0067] 2.1 Preparation of gold nanoparticle solution:

[0068] The gold nanoparticles used in the present invention have a size of 20 nm, and the preparation process is as follows:

[0069] Add a clean magnetic stir bar and 142.5 mL of ultrapure water to a three-necked round-bottom flask. Add 1.5 mL of 1% chloroauric acid and heat to gentle boiling simultaneously. Then quickly add 6 mL of 1% sodium citrate, continue stirring and maintain gentle boiling. The solution color immediately turns blue. Continue heating and stirring. The solution color changes from blue to purple and finally to red. After the solution color stabilizes and no longer changes, continue heating and stirring for 10 min. Then continue stirring until the solution cools to room temperature. Transfer it to a centrifuge tube, wrap it with tin foil to avoid light, and store at 4 °C.

[0070] 2.2 Probe sequence selection

[0071] First, the probe sequence must avoid overlapping or complementary with the primer sequence to avoid affecting the primer amplification efficiency. The length is between 15 - 21 bp, the GC% is between 40% - 60%, and the Tm value is between 50 °C - 60 °C. Based on the above considerations, the probe sequence was designed and modified on this basis to obtain the oligoA-tailed DNA sequence: 5’-CTAGCAGGTAATCTTGCCCACAAAAAA-3’ (SEQ ID No.7).

[0072] 2.3 Preparation of DNA-functionalized gold nanoprobe

[0073] Incubate the oligonucleotide chain in 2.2 with the gold nanoparticle solution prepared in 2.1. Modify the oligonucleotide chain onto the gold nanoparticles according to the molar concentration oligoA-tailed DNA:AuNPs = 600:1, thus completing the preparation of the gold nanoprobe, which can be used for subsequent amplification and color development experiments.

[0074] The specific steps for synthesizing the above gold nanoprobe are as follows:

[0075] 1) Dissolve 6 μL of 100 μM oligoA-tailed DNA in 59 μL of water, then mix it with 25 μL of 40 nM AuNPs and vortex to obtain a mixed solution;

[0076] 2) Add 10 μL of 200 mM NaNO3 to the above mixed solution and vortex to mix evenly;

[0077] 3) Add 700 μL of n-butanol to the mixed solution and quickly vortex to make the two phases mix evenly;

[0078] 4) Heat the mixed solution in a water bath at a heating temperature of 80 °C and a heating time of 30 min;

[0079] 5) Add 200 μL of TBE buffer to the heated mixed solution to separate the two phases;

[0080] 6) Reheat the mixed solution in a water bath at 80 °C for 30 min.

[0081] 7) Centrifuge to separate the two phases and take out the lower layer of oligoA-tailed DNA-AuNPs solution.

[0082] 8) Add an appropriate amount of ultrapure water and then centrifuge, discard the supernatant to obtain oligoA-tailed DNA-AuNPs. Measure the concentration for subsequent experiments.

[0083] III. The concentrations of each component for LAMP combined with AuNPs visualization detection are as follows:

[0084]

[0085] Figure 5 For Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and blank samples, the color development results in the amplification and color development system after adding different volumes of magnesium chloride are shown.

[0086] Specific operation steps: Place the amplification and color development systems containing samples of Atlantic salmon, rainbow trout and blank samples respectively in a constant temperature water bath at 60 °C for 40 min. After taking them out, add 4 μL of 5 μM magnesium chloride, and obvious color changes can be observed within 5 min. Among them, red represents Atlantic salmon and blue represents rainbow trout.

[0087] The primers, detection methods and applications for rapid visual identification of Atlantic salmon and rainbow trout by gold nanoparticles combined with loop-mediated isothermal amplification provided by the present invention are introduced in detail above. The above specific embodiments cannot be used to limit the protection scope of the present invention. For those skilled in the art of this technology, any alternative improvement or transformation made to the embodiments of the present invention falls within the protection scope of the present invention.

[0088] Where the present invention is not described in detail are all well-known technologies to those skilled in the art of this technology.

Claims

1. A primer set for rapid visual identification of Atlantic salmon and rainbow trout by gold nanoparticle combined loop-mediated isothermal amplification, characterized in that, It includes an outer primer pair F3 and B3, an inner primer pair FIP and BIP, a loop primer pair LF and LB, and a nanogold probe; The sequences of the outer primer pair F3 and B3 are shown as SEQ ID No.1 and SEQ ID No.2; The sequences of the inner primer pair FIP and BIP are shown as SEQ ID No.3 and SEQ ID No.4; The sequences of the loop primer pair LF and LB are shown as SEQ ID No.5 and SEQ ID No.6; The DNA sequence of the nanogold probe is shown as SEQ ID No.

7.

2. The primer set for rapid visual identification of Atlantic salmon and rainbow trout by gold nanoparticle combined loop-mediated isothermal amplification according to claim 1, characterized in that, The nanogold probe is prepared by mixing oligoA-tailed DNA and nanogold at a molar ratio of not less than 400:1; the size of the nanogold is 10 - 50 nm.

3. A kit for rapid visual identification of Atlantic salmon and rainbow trout by gold nanoparticle combined loop-mediated isothermal amplification, characterized in that, It includes the primer group as described in claim 1 or 2.

4. A detection method for rapid visual identification of Atlantic salmon and rainbow trout by combining gold nanoparticles with loop-mediated isothermal amplification, characterized in that, It includes the following operation steps: (1) Construct an amplification system The amplification system includes the primer group as described in claim 1 or 2, as well as Buffer, dNTP, MgSO4, Bst DNA polymerase, water, and a template; (2) Place the above amplification system in a 65°C constant temperature water bath for 60 min. After taking it out, add a certain volume of 5 μM MgCl2 solution. After 5 min, identify the detection result according to the color change: when the color always remains red, it indicates that the identified species or meat product is Atlantic salmon; if the color changes from red to blue, it indicates that the identified species or meat product is rainbow trout.

5. The application of the primer group as described in claim 1 or 2 and the kit as described in claim 3 in identifying Atlantic salmon and rainbow trout.

6. The application according to claim 5, wherein During identification, the color development time of 5 min can distinguish between Atlantic salmon and rainbow trout.

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