Specific primers and fluorescent quantitative PCR method for detecting ingredients of Peru anchovy in feed

Abstract

The present application belongs to the technical field of feedstuff raw material detection, and particularly relates to specific primers and probes for detecting ingredients of Thryssa afuerae in feedstuff and a fluorescent quantitative PCR method. The present application takes Thryssa afuerae mitochondrial COI gene as a specific detection target, and designs exclusive primer pairs and a TaqMan probe. The sequence of the upstream primer is shown as SEQ ID NO. 1, the sequence of the downstream primer is shown as SEQ ID NO. 2, and the sequence of the probe is shown as SEQ ID NO. 3. The probe is labeled with a CY5 fluorescent reporter group at the 5' end and a BHQ2 quencher group at the 3' end. The present application is 100% specific to Thryssa afuerae, can distinguish Thryssa afuerae from related species, and has a minimum detection limit and a quantitative lower limit of 0.1%, a quantitative range of 0.1% to 100%, excellent detection precision and accuracy, and can quickly realize qualitative identification and accurate quantification of ingredients of Thryssa afuerae in feedstuff, and is suitable for the quality control and market supervision needs of the feedstuff industry.

Description

Technical Field

[0001] This invention belongs to the field of feed ingredient detection technology, specifically relating to a specific primer and a real-time quantitative PCR method for detecting Peruvian anchovy-derived components in feed. Background Technology

[0002] The feed industry is the core support of modern livestock and aquaculture. Animal-derived protein raw materials are an indispensable core component of compound feed, and their types, sources, and contents directly determine the nutritional quality, feeding effects, and economic benefits of the feed. Among them, Peruvian anchovies (… Engraulis ringens As one of the world's most heavily fished commercial fish species, the fishmeal made from it has significant advantages such as high crude protein content, balanced amino acid composition, rich essential amino acid content, and high digestibility. It is the preferred high-quality animal protein raw material for high-end aquatic feed and young livestock and poultry feed, and its market price and commercial value are far higher than those of ordinary freshwater fishmeal and other low-value marine fishmeal substitutes.

[0003] Establishing accurate, efficient, and standardized methods for qualitative identification and quantitative detection of Peruvian anchovy-derived components in feed is a core technical requirement for combating raw material adulteration, controlling feed quality, and regulating market order. It is also a key technical challenge that the industry urgently needs to solve.

[0004] Currently, various technical methods have been developed and applied in the industry for the detection of animal-derived components in feed. The mainstream methods mainly include microscopic examination, near-infrared spectroscopy, conventional polymerase chain reaction (PCR) and real-time quantitative PCR. Each method has different technical limitations in practical applications, and in particular, they cannot fully meet the needs of accurate detection of Peruvian anchovy-derived components in feed.

[0005] Microscopic examination is a traditional method for detecting animal-derived components in feed. Its core principle is to identify animal-derived components from different sources by observing the morphological characteristics of animal tissues in a sample under a microscope. However, this method has significant technical drawbacks: First, the test results are highly dependent on the professional skills and practical experience of the testers, with significant subjective influence. For feed samples that have undergone deep processing such as high temperature, high pressure, and extrusion, their tissue morphology is severely damaged, making it easy to miss or misjudge, resulting in a high overall false negative rate. Second, this method can only achieve qualitative screening and cannot accurately quantify the target source component, nor can it determine the actual content of Peruvian anchovy meal in the sample. Third, this method cannot distinguish source components from closely related species. It has no effective ability to differentiate between Peruvian anchovy and fishmeal from closely related species such as European anchovy and Japanese anchovy, and is completely unable to deal with adulteration by passing off low-value fishmeal as Peruvian anchovy meal.

[0006] Near-infrared spectroscopy, based on the differences in near-infrared spectral characteristics of raw materials from different species, enables rapid analysis of target components in samples through a pre-constructed, proprietary calibration model. However, this method has significant shortcomings in the detection of Peruvian anchovy-derived components: Firstly, the detection accuracy of this method is highly dependent on the coverage and accuracy of the calibration model. Feed product formulations are complex, with numerous substrate types, and different processing techniques can significantly interfere with the spectral characteristics of the samples. The detection results are easily affected by feed substrates, sample morphology, processing methods, and environmental factors, resulting in poor stability and repeatability. Secondly, this method lacks specificity. It cannot effectively distinguish closely related species with small differences in gene sequences and composition, making it difficult to accurately identify Peruvian anchovy from closely related fishmeal species, thus failing to meet the practical needs for precise identification.

[0007] Conventional PCR, targeting species-specific gene sequences, achieves qualitative detection of target-derived components through PCR amplification, offering improved specificity and sensitivity compared to microscopy and near-infrared spectroscopy. However, this method only provides qualitative identification and cannot accurately quantify the content of target-derived components in samples. It also cannot determine the actual amount of Peruvian anchovy meal added to feed, making it difficult to support quality grading of feed products and screening for high-quality feed, and ultimately failing to meet the industry's need for end-to-end quality control of Peruvian anchovy-derived components.

[0008] Real-time quantitative PCR (qPCR) technology, with its advantages of high specificity, high sensitivity, quantification, good repeatability, and automated detection, has become the mainstream technology for detecting animal-derived components in feed. This technology is mainly based on TaqMan fluorescent probes or fluorescent dyes, achieving qualitative and quantitative analysis of target genes by monitoring changes in fluorescence signals during PCR amplification in real time. However, currently, there is no standardized and precisely quantifiable qPCR detection method for Peruvian anchovy-derived components in feed. Existing research and technologies can only achieve qualitative detection or preliminary quantification of Peruvian anchovy components in pure fishmeal matrices, which cannot be adapted to the detection scenarios of complex matrices in formulated feeds. In complex feed matrices, it is easily affected by non-target components, resulting in a significant decrease in quantitative accuracy. Furthermore, existing primer and probe systems cannot effectively distinguish between Peruvian anchovies and closely related species such as European and Japanese anchovies, easily leading to cross-reactions and false positive results. This makes it difficult to accurately identify low-value adulteration with fishmeal of the same genus, and fails to meet the practical needs of high-end feed for precise identification, content determination, and quality screening.

[0009] In summary, existing technologies for detecting animal-derived components in feed cannot simultaneously achieve highly specific qualitative identification and highly accurate quantitative detection of Peruvian anchovy-derived components in complex feed matrices. These technologies fail to effectively address the core problem of adulteration and counterfeiting of Peruvian anchovy meal in the industry. There is an urgent need to develop a fluorescence quantitative PCR detection method for Peruvian anchovy-derived components that is highly specific, sensitive, quantitatively accurate, resistant to matrix interference, and capable of distinguishing closely related species of the genus *Anchovy*. This method would provide a standardized technical means for detecting Peruvian anchovy-derived components in feed and fill relevant technological gaps. Summary of the Invention

[0010] The purpose of this invention is to provide a specific primer and a real-time quantitative PCR method for detecting Peruvian anchovy-derived components in feed, which completely solves the industry pain point that existing detection methods cannot accurately identify low-value fishmeal from the same genus that is being passed off as Peruvian anchovy meal, and provides a reliable technical basis for the accurate identification of Peruvian anchovy-derived components in feed.

[0011] The objective of this invention is achieved through the following technical solution: This invention provides a specific primer-probe composition for detecting Peruvian anchovy-derived components in feed, comprising an upstream primer, a downstream primer, and a TaqMan fluorescent probe; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.3.

[0012] Furthermore, the TaqMan fluorescent probe is labeled with a CY5 fluorescent reporter group at its 5' end and a BHQ2 quencher group at its 3' end.

[0013] The present invention also provides a real-time quantitative PCR method for detecting Peruvian anchovy-derived components in feed, comprising real-time quantitative PCR amplification of genomic DNA of the feed sample to be tested using the specific primer and probe composition described above.

[0014] Furthermore, the quantitative real-time PCR amplification uses a total reaction system of 20 μL, with the following components and amounts: 10 μL 2×PCR reaction buffer, 0.6 μL 10 μmol / L upstream primer, 0.6 μL 10 μmol / L downstream primer, 0.2 μL 10 μmol / L TaqMan fluorescent probe, 7.6 μL RNase-free water, and 1 μL genomic DNA template of the sample to be tested.

[0015] Furthermore, the procedure for the quantitative PCR amplification is as follows: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 10 s; annealing and extension at 60℃ for 30 s, and collection of fluorescence signal in the CY5 channel, for a total of 40 cycles.

[0016] Furthermore, the method for extracting genomic DNA from the feed sample to be tested is as follows: after grinding the feed sample to be tested evenly, genomic DNA is extracted using the metal bath method of a DNA / RNA co-extraction and purification kit, and A is screened. 260 / A 280 Genomic DNA with a ratio of 1.8 to 1.9 was diluted to 100 ng / μL and used as a template for PCR amplification.

[0017] Furthermore, it also includes a qualitative result determination step: if the amplification Ct value of the sample to be tested is <35, it is determined that Peruvian anchovy-derived components are detected; if the amplification Ct value of the sample to be tested is ≥35, it is determined that Peruvian anchovy-derived components are not detected.

[0018] Furthermore, it also includes a quantitative detection step: a quantitative standard curve is constructed using Peruvian anchovy meal standards with gradient contents, and the amplified Ct value of the sample to be tested is substituted into the quantitative standard curve to calculate the mass fraction of Peruvian anchovy-derived components in the sample to be tested.

[0019] Furthermore, the linear regression equation of the quantitative standard curve is Y = -3.2867X + 30.848, where Y is the Ct value, X is the logarithm of the mass fraction of Peruvian anchovy meal, and the correlation coefficient R of the standard curve is... 2 =0.995, with a quantitative range of 0.1% to 100%.

[0020] Furthermore, the feed samples to be tested include fishmeal raw materials, aquatic compound feed, and livestock and poultry compound feed.

[0021] The beneficial effects of this invention are as follows: This invention addresses the core shortcomings of existing Peruvian anchovy-derived component detection technologies, which cannot distinguish between closely related species of the genus *Anchovy* and are prone to cross-reactivity leading to false positive results. Using the *Anchovy* mitochondrial cytochrome c oxidase subunit I gene as the detection target, this invention screens for conserved specific regions unique to Peruvian anchovies that have no homology with closely related species or other common feed ingredient species. A specific primer and probe combination is designed based on this combination, which exhibits 100% species specificity for Peruvian anchovies, producing specific amplification only in *Anchovy* samples. Closely related species to European and Japanese anchovies, this product showed no cross-reactivity with other aquatic raw materials such as bass, yellow catfish, and mackerel, as well as livestock and poultry raw materials such as pigs, cattle, sheep, chickens, and ducks, aquaculture species such as whiteleg shrimp, and blank feed substrates that do not contain Peruvian anchovies. This eliminates false positive results at the source of the detection system and completely solves the industry pain point that existing detection methods cannot accurately identify low-value fishmeal that is being passed off as Peruvian anchovy meal. This provides a reliable technical foundation for the accurate identification of Peruvian anchovy-derived components in feed.

[0022] The quantitative real-time PCR detection method established in this invention possesses excellent detection sensitivity and quantitative performance. Its limit of detection (LOD) and limit of quantitation (LOQ) can both reach 0.1% by mass fraction, enabling stable detection of trace amounts of Peruvian anchovy-derived components in feed. This effectively avoids the problem of missed detection in low-dose adulterated samples found in existing technologies and can cover most adulteration scenarios involving trace amounts in the market. The quantitative detection range of this method is 0.1% to 100%. Within this range, the logarithm of the Peruvian anchovy meal mass fraction and the detected Ct value show a very strong linear correlation, with a correlation coefficient Rt of the fitted standard curve. 2 The amplification efficiency can reach 0.995, meeting the optimal standard for quantitative PCR detection. For the detection of Peruvian anchovy-derived components in complex feed matrices, the quantitative relative error is less than 5%, which is far lower than the error level of conventional detection methods in the industry. At the same time, the intra-batch and inter-batch detection coefficients of variation of this method are both less than 0.5%. The detection results are not affected by external factors such as the detection batch or the operator. It has excellent detection precision and result stability, and completely solves the technical defects of existing technologies that can only achieve quantification in pure fishmeal matrices and whose quantification accuracy drops significantly in complex compound feed matrices. It realizes the accurate quantification of Peruvian anchovy-derived components in compound feed.

[0023] The detection method established in this invention has strong practical application adaptability and scalability. The reagents, consumables, and instruments used in the detection process are all commercially available conventional products in the field of molecular biology detection. There is no need to configure special equipment. The operation process is standardized and controllable, and it does not rely on the long-term practical experience of the testing personnel. It can effectively reduce the threshold of detection operation. It is suitable for standardized laboratory testing in professional testing institutions, and can also meet the daily batch testing needs of feed production enterprises for raw material quality control and finished product batch testing. The method has strong adaptability to the matrix of the test sample and can be adapted to a variety of test matrices such as fishmeal raw materials, aquatic compound feeds with different formulations, and livestock and poultry compound feeds. It is not affected by the processing technology and formulation of feed. Whether it is finished compound feed that has undergone high temperature, high pressure, and extrusion treatment, or fishmeal raw materials from different sources, stable and accurate detection can be achieved. It can comprehensively cover the detection needs of Peruvian anchovy-derived components in the entire feed production chain.

[0024] This invention fills the technological gap in the current feed industry where there is no standardized and accurately quantifiable fluorescence quantitative PCR detection method for Peruvian anchovy-derived components. It integrates highly specific qualitative identification with high-precision quantitative detection, enabling the determination of the presence and content of Peruvian anchovy-derived components in a single test. This allows for the rapid identification of illegal activities such as the substitution of low-value fishmeal with Peruvian anchovy meal in fishmeal raw materials and finished feeds, providing precise technical support for market supervision. It also provides reliable testing basis for raw material quality control and finished product quality grading for feed production enterprises. Furthermore, it assists aquaculture enterprises in the rapid screening of high-quality feed, effectively protecting the legitimate rights and interests of feed production enterprises and aquaculture practitioners. This invention has significant practical application value for regulating the feed raw material market order, improving feed product quality, and promoting the healthy development of the livestock and aquaculture industries. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Amplification curves were used to verify the specificity of the primers and probes. Figure 2 Graphs of real-time PCR amplification of Peruvian anchovies at different DNA concentration gradients; Figure 3 Graphs of real-time quantitative PCR amplification of Peruvian anchovy meal samples with different contents; Figure 4 Quantitative standard curve for Peruvian anchovy meal gradient standards; Figure 5 This is an amplification curve for testing actual feed samples. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional molecular biology experimental methods in the field; the reagents, consumables, and instruments used are all products that can be obtained through commercial channels; among them, genomic DNA extraction was performed using a DNA / RNA co-extraction and purification kit (metal bath method) produced by Jiangsu Liezhen Biotechnology Co., Ltd., and all primers and probes were synthesized and purified by HPLC by a professional biosynthesis company.

[0033] In this invention, the qualitative judgment criteria for fluorescence quantitative PCR detection are uniformly defined as follows: if the Ct value of the test sample is <35, it is determined that Peruvian anchovy-derived components are detected; if the Ct value of the test sample is ≥35, it is determined that Peruvian anchovy-derived components are not detected.

[0034] Example 1: Design and Specificity Verification Experiment of Peruvian Anchovy-Specific Primers and Probes This embodiment is used to verify the species specificity of the primers and probes designed in this invention for Peruvian anchovies, as well as the lack of cross-reactivity with closely related species, other common feed ingredient species, and blank feed matrix.

[0035] 1. Experimental Materials and Instruments 1.1 Experimental Samples Positive sample: Peruvian anchovy (Engraulis ringens) standard; Negative control samples: closely related species (European anchovy, Japanese anchovy), other common feed ingredient species (bass, yellow catfish, mackerel, sardine, cod, pig, cattle, sheep, chicken, duck, whiteleg shrimp), and blank compound feed without Peruvian anchovy-derived components; 1.2 Experimental Reagents DNA / RNA co-extraction and purification kit (metal bath method), 2×PCR reaction buffer, Taq DNA polymerase, RNase-free water. 1.3 Experimental Apparatus Real-time quantitative PCR instrument, electronic analytical balance, high-throughput tissue homogenizer, ultraviolet spectrophotometer, constant temperature water bath, high-speed centrifuge.

[0036] 2. Experimental Methods 2.1 Specific target sequence screening and primer / probe design Using the mitochondrial cytochrome c oxidase subunit I (COI) gene of anchovies as a specific detection target, the full-length COI gene sequences of anchovies, closely related species of the above genus, and other non-target species were downloaded from the GenBank database of the National Center for Biotechnology Information (NCBI). ClustalX software was used to perform multiple sequence alignment on all sequences to screen out conserved specific sequence segments that are unique to anchovies and have no homology with closely related species and other non-target species.

[0037] Using PrimerExpress 3.0 software, TaqMan quantitative PCR primers and probes were designed for the specific regions identified above. The design parameters were set as follows: primer length 18–24 bp, Tm value 58–62℃; probe length 20 bp, Tm value 65–68℃. Simultaneously, non-specific amplification risks such as primer dimers and hairpin structures were strictly excluded. The final specific primer and probe sequences are as follows: Upstream primer (SEQ ID NO.1): 5'-TGGGCTCATCATATGTTTACAGTGG-3' Downstream primer (SEQ ID NO.2): 5'-CGCCCAAAGCATAGGAGT-3' TaqMan probe (SEQ ID NO.3): 5'-CY5-CCCGTGCAAAGTAGCGAGT-BHQ2-3', wherein the 5' end of the probe is labeled with a CY5 fluorescent reporter group and the 3' end is labeled with a BHQ2 quencher group.

[0038] 2.2 Genomic DNA Extraction from Samples Take all the above experimental samples, grind them into a uniform powder, and extract genomic DNA from each sample according to the instructions of the DNA / RNA co-extraction and purification kit (metal bath method); use a UV spectrophotometer to detect the purity and concentration of the extracted genomic DNA, and screen for A... 260 / A 280 DNA samples with a ratio between 1.8 and 1.9 should be uniformly diluted to 100 ng / μL and stored at -20℃ for later use.

[0039] 2.3 Quantitative Real-Time PCR Reaction System and Amplification Procedure The total reaction volume was 20 μL, and the amounts of each component added were as follows: 10 μL 2×PCR reaction buffer, 0.6 μL 10 μmol / L upstream primer, 0.6 μL 10 μmol / L downstream primer, 0.2 μL 10 μmol / L fluorescent probe, 7.6 μL RNase-free water, and 1 μL diluted DNA template.

[0040] Simultaneously, a template-free blank control and a negative control without Peruvian anchovy DNA were set up, and all samples were configured with 3 technical replicates.

[0041] The quantitative real-time PCR amplification program was set as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s; 60℃ annealing extension for 30 s. Simultaneously, the fluorescence signal of the CY5 channel was collected during the annealing extension stage. A total of 40 cycles were set.

[0042] 2.4 Result Determination Based on the aforementioned unified qualitative judgment criteria, the specificity of primers and probes is determined according to the amplification curves and Ct values ​​of each sample.

[0043] 3. Experimental Results and Analysis The amplification results of this embodiment are as follows: Figure 1 As shown, only the Peruvian anchovy positive sample showed a typical S-shaped specific amplification curve with a Ct value < 35, and was judged as a positive detection; while the closely related species European anchovy, Japanese anchovy, as well as all other non-target species samples, blank feed samples, blank controls, and negative controls, did not show specific amplification curves and no effective Ct value was detected, and were judged as negative.

[0044] The results show that the primers and probes designed in this invention have 100% species specificity for Peruvian anchovies and show no cross-reactivity with closely related species, other common feed ingredient species, or blank feed matrix. They can accurately distinguish Peruvian anchovies from closely related species of the genus Anchovy and are fully applicable to the qualitative and quantitative detection of Peruvian anchovy-derived components in feed.

[0045] Example 2: Sensitivity Experiment of the Quantitative Real-Time PCR Detection Method of the Present Invention This embodiment is used to verify the limit of detection and sensitivity of the detection method of the present invention for Peruvian anchovy-derived DNA.

[0046] 1. Experimental Materials and Instruments 1.1 Experimental Samples The Peruvian anchovy genomic DNA sample prepared in Example 1 had a concentration of 100 ng / μL; 1.2 Experimental Reagents RNase-free water, and the real-time PCR reagent described in Example 1; 1.3 Experimental Apparatus The same real-time fluorescence quantitative PCR instrument and matching consumables as described in Example 1.

[0047] 2. Experimental Methods Using RNase-free water, genomic DNA samples of Peruvian anchovies at a concentration of 100 ng / μL were serially diluted 10-fold to prepare DNA templates at five concentration gradients: 100 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, and 0.01 ng / μL.

[0048] Using DNA diluted at each concentration gradient as templates, amplification was performed using the primers, probes, real-time PCR reaction system, and amplification program described in Example 1. Three technical replicates were set up for each concentration gradient, and a blank control was set up simultaneously. The Ct values ​​and amplification curves of each sample were recorded.

[0049] 3. Experimental Results and Analysis Table 1 shows the Ct values ​​of real-time PCR amplification of Peruvian anchovy genomic DNA at different concentration gradients, and the amplification curves are shown in Table 1. Figure 2 As shown.

[0050] Table 1. Ct values ​​of fluorescent PCR amplification of Peruvian anchovies at different DNA concentration gradients.

[0051] Note: "N / A" indicates not detected, the same applies below.

[0052] The results showed that the Ct value of the Peruvian anchovy DNA template increased regularly with decreasing template concentration, exhibiting a good linear relationship. Using Ct < 35 as the criterion for effective amplification, samples in the concentration range of 100 ng / μL to 0.1 ng / μL all showed stable specific amplification with Ct values ​​< 35, and the reproducibility of the three technical replicates was good. For samples with a concentration of 0.01 ng / μL, the Ct value was > 35, and no stable effective amplification signal was observed.

[0053] Using the logarithm of template concentration as the x-axis and the corresponding average Ct value as the y-axis, the fitted sensitivity standard curve is Y = -3.734X + 31.798, with a correlation coefficient R² = 0.997, showing excellent linearity.

[0054] In summary, the fluorescence quantitative PCR detection method established in this invention has a detection limit of 0.1 ng / μL for Peruvian anchovy genomic DNA, can stably detect trace amounts of Peruvian anchovy-derived DNA, and has excellent detection sensitivity, which can meet the detection requirements for trace Peruvian anchovy-derived components in feed.

[0055] Example 3: Construction of quantitative standard curve and verification of accuracy and precision. This embodiment is used to construct a quantitative standard curve for the content of Peruvian anchovy meal in feed matrix and to verify the quantitative accuracy, detection precision and stability of the method of the present invention in complex feed matrix.

[0056] 1. Experimental Materials and Instruments 1.1 Experimental Samples 100% pure Peruvian anchovy meal, a basic formulated feed free of Peruvian anchovy-derived ingredients; 1.2 Experimental Reagents and Instruments The same DNA extraction reagent, real-time PCR reagent, and experimental instruments as described in Example 1.

[0057] 2. Experimental Methods 2.1 Preparation of gradient standards Using 100% pure Peruvian anchovy meal as raw material, it was precisely weighed and thoroughly ground and mixed with a basic compound feed without Peruvian anchovy at a preset mass ratio to prepare standard samples of Peruvian anchovy meal with 6 mass fraction gradients, namely 0.1%, 1%, 10%, 20%, 30% and 100%, and 3 biological replicates were set up for each mass fraction gradient.

[0058] 2.2 Genomic DNA Extraction and Quantitative PCR Amplification of Standard Samples Following the genomic DNA extraction method described in Example 1, genomic DNA was extracted from the above-mentioned gradient standards, and the DNA purity A was detected. 260 / A280 Dilute the sample to 100 ng / μL between 1.8 and 1.9 for later use. Amplify the sample using the fluorescence quantitative PCR reaction system and amplification program described in Example 1. Set up three technical replicates for each sample and record the amplification curves and average Ct values ​​for each gradient sample.

[0059] 2.3 Fitting of the Quantitative Standard Curve Using the logarithm of the mass fraction of Peruvian anchovy meal (lgC) as the abscissa (X) and the average Ct value of the corresponding gradient samples as the ordinate (Y), linear regression was performed to construct a quantitative standard curve, obtain the linear regression equation and correlation coefficient R², and calculate the amplification efficiency.

[0060] 2.4 Quantitative Accuracy Validation Blank basic feed, which was confirmed to contain no Peruvian anchovy-derived components, was precisely weighed and mixed with 100% Peruvian anchovy pure fishmeal to prepare Peruvian anchovy feed verification samples with theoretical mass fractions of 5% and 40%, respectively. Three parallel samples were set up for each mass fraction gradient.

[0061] Genomic DNA was extracted from the verification samples according to the method described in Example 1. Amplification was performed using the fluorescence quantitative PCR reaction system and amplification program of the present invention, and the average Ct value of each sample was recorded. The measured Ct value was substituted into the linear regression equation of the quantitative standard curve constructed above to calculate the measured mass fraction of Peruvian anchovy meal in the sample, and the quantitative relative error was calculated to verify the quantitative accuracy of the method.

[0062] 2.5 Precision Verification The above-mentioned standards at three gradients of 0.1%, 10%, and 100% were selected, and intra-batch repeatability testing (6 technical replicates were set in the same amplification experiment) and inter-batch repeatability testing (one amplification experiment was performed each day for 3 consecutive working days, with 3 technical replicates set each time) were carried out. The coefficient of variation of the intra-batch and inter-batch test results were calculated to verify the detection precision and stability of the method.

[0063] 3. Experimental Results and Analysis 3.1 Amplification results of gradient standards Amplification curves of Peruvian anchovy meal standards with different mass fraction gradients are shown below. Figure 3 As shown, all gradient samples exhibited stable, specific S-shaped amplification curves. The Ct value increased systematically with decreasing Peruvian anchovy meal mass fraction. Even at a mass fraction of 0.1%, stable specific amplification was still achieved, with a Ct value < 35. The average Ct values ​​for each gradient sample are shown in Table 2.

[0064] Table 2. Ct values ​​of different mass fractions of Peruvian anchovy meal amplified by fluorescent PCR.

[0065] 3.2 Results of Quantitative Standard Curve Fitting The quantitative standard curve obtained by fitting the logarithm of the mass fraction of Peruvian anchovy meal to the x-axis and the mean Ct value to the y-axis is shown below. Figure 4 As shown, the linear regression equation is: Y = -3.2867X + 30.848, the correlation coefficient R² = 0.995, and the amplification efficiency is 101.5%.

[0066] The results showed that, within the mass fraction range of 0.1% to 100%, there was a strong linear correlation between the logarithm of the mass fraction of Peruvian anchovy meal and the average Ct value obtained. The detection method established in this invention has a wide quantitative range, which can cover the conventional addition range of Peruvian anchovy meal in feed, and the lowest quantitative limit can reach 0.1%.

[0067] 3.3 Quantitative accuracy verification results The accuracy verification results are as follows: For the verification sample with a theoretical mass fraction of 5%, the average Ct value is 28.56. Substituting this value into the standard curve equation, the measured average mass fraction is 4.81%, and the quantitative relative error is 3.80%. For the verification sample with a theoretical mass fraction of 40%, the average Ct value is 25.59. Substituting this value into the standard curve equation, the measured average mass fraction is 39.78%, and the quantitative relative error is 0.55%.

[0068] The quantitative relative errors for both gradient validation samples were less than 5%, far below the error levels of conventional industry testing methods. The results indicate that the detection method of this invention can effectively resist interference from complex feed matrices, achieving accurate quantification of Peruvian anchovy-derived components even in formulated feed matrices, demonstrating excellent quantitative accuracy and fully meeting the needs of practical testing.

[0069] 3.4 Precision Validation Results Precision testing results showed that the intra-batch coefficients of variation for the three graded standards (0.1%, 10%, and 100%) were all <0.5%, and the inter-batch coefficients of variation were also <0.5%. These results indicate that the detection method of this invention has excellent precision and repeatability, and the test results are stable and reliable, unaffected by factors such as testing time or operator skill. It is suitable for routine laboratory testing and batch quality control testing in enterprises.

[0070] Example 4: Detection and Application Experiment of Actual Fishmeal and Commercially Available Feed Samples This embodiment is used to verify the applicability and detection effect of the detection method of the present invention on fishmeal from different sources and commercially available feed samples of different types in actual production scenarios.

[0071] 1. Experimental Materials and Instruments 1.1 Sample to be tested Fish meal samples to be tested (5 types): domestic freshwater fish meal, Peruvian anchovy meal, Japanese fish meal, domestic fish meal, and imported super fish meal; Five commercially available compound feed samples were tested: sea bass feed, whiteleg shrimp feed, yellow catfish feed, chicken feed, and grouper feed. 1.2 Experimental Reagents and Instruments The same DNA extraction reagent, real-time PCR reagent, and experimental instruments as described in Example 1.

[0072] 2. Experimental Methods Following the genomic DNA extraction method described in Example 1, genomic DNA was extracted from all the above-mentioned samples to be tested, and the DNA purity A was detected. 260 / A 280 The concentration was kept between 1.8 and 1.9, and then diluted to 100 ng / μL for later use. The fluorescence quantitative PCR reaction system and amplification program described in Example 1 were used for detection. Three technical replicates were set up for each sample to be tested, and the average Ct value and amplification curve of each sample were recorded.

[0073] The presence or absence of Peruvian anchovy-derived components in the sample was determined according to the aforementioned unified qualitative judgment criteria. For samples that tested positive, the measured average Ct value was substituted into the linear regression equation Y=-3.2867X+30.848 of the quantitative standard curve constructed in Example 3 to calculate the actual mass fraction of Peruvian anchovy-derived components in the sample.

[0074] 3. Experimental Results and Analysis The amplification curve of this embodiment is as follows: Figure 5 As shown in Table 3, the test results of the 10 samples are as follows.

[0075] Table 3. Test results of fishmeal and commercially available feed samples from different sources.

[0076] The results show that the fluorescence quantitative PCR detection method established in this invention has strong matrix adaptability and can be adapted to a variety of complex detection matrices such as freshwater fishmeal, imported fishmeal, aquatic compound feed, and livestock and poultry compound feed. It can accurately distinguish Peruvian anchovy meal from other common fishmeal, effectively identify adulteration and counterfeiting of fishmeal, and accurately quantify the actual content of Peruvian anchovy-derived components in the sample based on the constructed standard curve.

[0077] The detection method of this invention provides stable detection results, clear judgment criteria, and standardized operating procedures, fully meeting the application needs of qualitative identification, content determination, feed quality control, and rapid screening of high-quality feed derived from Peruvian anchovies in actual production. It has excellent practical detection value and industry applicability.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A specific primer-probe composition for detecting Peruvian anchovy-derived components in feed, characterized in that, It consists of an upstream primer, a downstream primer, and a TaqMan fluorescent probe; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.

3.

2. The specific primer-probe composition according to claim 1, characterized in that, The TaqMan fluorescent probe is labeled with a CY5 fluorescent reporter group at its 5' end and a BHQ2 quencher group at its 3' end.

3. A real-time quantitative PCR method for detecting Peruvian anchovy-derived components in feed, characterized in that, This includes using the specific primer and probe composition according to any one of claims 1-2 to perform real-time PCR amplification of the genomic DNA of the feed sample to be tested.

4. The real-time PCR method according to claim 3, characterized in that, The quantitative real-time PCR amplification used a total reaction system of 20 μL, with the following components and amounts: 10 μL 2×PCR reaction buffer, 0.6 μL 10 μmol / L upstream primer, 0.6 μL 10 μmol / L downstream primer, 0.2 μL 10 μmol / L TaqMan fluorescent probe, 7.6 μL RNase-free water, and 1 μL genomic DNA template from the sample to be tested.

5. The real-time PCR method according to claim 3, characterized in that, The procedure for quantitative real-time PCR amplification is as follows: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 10 s; annealing and extension at 60℃ for 30 s, and collection of fluorescence signal in the CY5 channel, for a total of 40 cycles.

6. The real-time PCR method according to claim 3, characterized in that, The method for extracting genomic DNA from the feed sample to be tested is as follows: After grinding the feed sample to be tested evenly, genomic DNA is extracted using the metal bath method of a DNA / RNA co-extraction and purification kit, and A is screened. 260 / A 280 Genomic DNA with a ratio of 1.8 to 1.9 was diluted to 100 ng / μL and used as a template for PCR amplification.

7. The real-time PCR method according to claim 3, characterized in that, It also includes a qualitative result determination step: if the amplification Ct value of the sample to be tested is <35, it is determined that Peruvian anchovy-derived components are detected; if the amplification Ct value of the sample to be tested is ≥35, it is determined that Peruvian anchovy-derived components are not detected.

8. The real-time PCR method according to claim 3, characterized in that, It also includes a quantitative detection step: a quantitative standard curve is constructed using Peruvian anchovy meal standards with gradient contents, and the amplified Ct value of the sample to be tested is substituted into the quantitative standard curve to calculate the mass fraction of Peruvian anchovy-derived components in the sample to be tested.

9. The real-time PCR method according to claim 8, characterized in that, The linear regression equation for the quantitative standard curve is Y = -3.2867X + 30.848, where Y is the Ct value, X is the logarithm of the mass fraction of Peruvian anchovy meal, and the correlation coefficient R of the standard curve is... 2 =0.995, with a quantitative range of 0.1% to 100%.

10. The real-time PCR method according to claim 3, characterized in that, The feed samples to be tested include fishmeal raw materials, aquatic compound feed, and livestock and poultry compound feed.

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