Water environment MST primer performance evaluation method, device, equipment and medium

Abstract

The invention relates to a water environment MST primer performance evaluation method, device and equipment and a medium. The method comprises the following steps: acquiring a water environment sample; the water environment sample comprises a target pollution sample and a non-target microorganism sample; performing DNA extraction on the water environment sample, and performing concentration standardization treatment on the water environment sample to obtain a positive sample library and a negative sample library; respectively carrying out amplification reaction on a target primer and the positive sample library and the negative sample library to obtain amplification curve data and melting curve data; based on the melting curve data and the amplification curve data, obtaining a sensitivity index and a specificity index corresponding to the target primer; obtaining a primer performance result based on the sensitivity index, the specificity index and a circulation threshold value in the amplification curve data; the primer performance result is used for representing whether the primer performance reaches the standard or not. By adopting the method, the accuracy of primer performance evaluation can be improved, and the reliability of water environment MST detection is guaranteed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganism tracing, and particularly relates to a water environment MST primer performance evaluation method, device, equipment and medium. BACKGROUND

[0002] With the rapid development of the field of water environment microorganism source tracking (MST), an MST detection technology based on specific primers has emerged. This technology can accurately lock in target pollution sources such as human and livestock manure, and effectively solve the problem of strong ambiguity of traditional tracing methods by virtue of high targeting and high detection efficiency, and has become the core technology of current tracing analysis in water environment pollution prevention and control. The performance of the primer directly determines the application effect of the technology, thus leading to the traditional water environment MST primer performance evaluation method.

[0003] In the traditional technology, when evaluating the performance of the MST primer, only a small amount of single type of target pollution sample (such as only human manure sample) is collected, and after DNA extraction of the sample, PCR (Polymerase Chain Reaction) experiment is carried out without strict concentration standardization treatment. The sensitivity and specificity of the primer are judged only by observing whether the amplification band exists or not and roughly calculating the detection limit, and environmental background flora and other non-target microorganism samples are rarely included for interference verification.

[0004] However, the traditional evaluation method has the following problems: first, the sample coverage is narrow, and the consideration of various livestock manure samples and complex environmental background flora is lacking, making it difficult to simulate the complex situation of the actual water environment; second, the DNA concentration is not standardized, and the concentration difference between samples will lead to distortion of the PCR amplification result, affecting the accuracy of the data; third, the evaluation index is not comprehensive, and the melting temperature value of the melting curve and the cycle threshold value of the amplification curve and other key parameters are not combined, which cannot comprehensively and accurately evaluate the primer performance, and is easy to cause misjudgment of the primer performance, thereby affecting the reliability of subsequent water environment MST detection. SUMMARY

[0005] Therefore, it is necessary to provide a water environment MST primer performance evaluation method, device, equipment and medium capable of improving the evaluation of primer performance in view of the above technical problems.

[0006] In a first aspect, the application provides a water environment MST primer performance evaluation method, comprising:

[0007] obtaining a water environment sample; the water environment sample comprises a target pollution sample and a non-target microorganism sample;

[0008] extracting DNA from the water environment sample, and performing concentration standardization treatment on the water environment sample to obtain a positive sample library and a negative sample library;

[0009] The target primer is used for amplification reaction with the positive sample library and the negative sample library respectively to obtain amplification curve data and melting curve data; the amplification curve data includes a positive amplification curve and a negative amplification curve; the melting curve data includes a positive melting curve and a negative melting curve.

[0010] Based on the melting curve data and the amplification curve data, a sensitivity index and a specificity index corresponding to the target primer are obtained.

[0011] Based on the sensitivity index, the specificity index, and a cycle threshold in the amplification curve data, a primer performance result is obtained; the primer performance result is used to represent whether the primer performance meets the standard.

[0012] In one of the embodiments, DNA extraction is performed on the water environment sample, and concentration standardization processing is performed on the water environment sample to obtain the positive sample library and the negative sample library, including:

[0013] DNA extraction is performed on the target pollution sample to obtain a positive DNA template.

[0014] DNA extraction is performed on the non-target microorganism sample to obtain a negative DNA template.

[0015] Concentration standardization is performed on the positive DNA template and the negative DNA template to obtain the positive sample library and the negative sample library with uniform concentration.

[0016] In one of the embodiments, the target primer is used for amplification reaction with the positive sample library and the negative sample library respectively to obtain amplification curve data and melting curve data, including:

[0017] Based on a preset fluorescent quantitative PCR reaction system, the target primer is mixed with the positive sample library and the negative sample library respectively to obtain a positive mixed solution and a negative mixed solution;

[0018] The positive mixed solution and the negative mixed solution are subjected to amplification processing respectively to obtain positive amplification products and negative amplification products corresponding to the target primer;

[0019] Based on the positive amplification products and the negative amplification products, amplification curve data and melting curve data are obtained.

[0020] In one of the embodiments, after the positive mixed solution and the negative mixed solution are subjected to amplification processing respectively to obtain positive amplification products and negative amplification products corresponding to the target primer, the following steps are further included:

[0021] Sequencing is performed on the positive amplification products and the negative amplification products to obtain sequencing results.

[0022] The sequencing result is compared with a preset microbial genome database to obtain a comparison and verification result, and the comparison and verification result is used to represent the specificity of the target primer.

[0023] In one embodiment, based on the melting curve data and the amplification curve data, a sensitivity index and a specificity index corresponding to the target primer are obtained, including:

[0024] The cycle threshold of all positive samples is extracted from the positive amplification curve to obtain the total amount of host samples detected;

[0025] The number of positive samples with a cycle threshold less than a preset amplification threshold is selected to obtain the true positive host sample amount detected;

[0026] Based on the total amount of host samples detected and the true positive host sample amount detected, the sensitivity index is calculated using the following formula:

[0027]

[0028] Sensitivity index S = P / P sens P is the true positive host sample amount detected, and P total is the total amount of host samples detected.

[0029] The cycle threshold of all negative samples is extracted from the negative amplification curve to obtain the total amount of expected negative host samples;

[0030] The number of negative samples with a cycle threshold greater than a preset amplification threshold is selected to obtain the amount of negative host samples detected;

[0031] Based on the total amount of expected negative host samples and the amount of negative host samples detected, the specificity index is calculated using the following formula:

[0032]

[0033] Specificity index S = N / N spec N is the amount of negative host samples detected, and N total is the total amount of expected negative host samples.

[0034] In one embodiment, based on the sensitivity index, the specificity index, and the cycle threshold in the amplification curve data, a primer performance result is obtained, including:

[0035] Multiple repeated experiments are performed on the positive amplification curve to obtain multiple sets of cycle threshold values;

[0036] The multiple sets of cycle threshold values are averaged to obtain a set of average values;

[0037] The cycle threshold standard deviation is calculated using the following formula:

[0038]

[0039] Where, σ Ct The standard deviation of the cyclic threshold. Let μ be the average number of cycles when the detection threshold is reached in the i-th repeated experiment, μ be the overall mean of the set of averages, and n be the number of repeated experiments.

[0040] Based on sensitivity indicators, specificity indicators, and cycle threshold standard deviation, combined with preset achievement rules, primer performance results are obtained.

[0041] In one embodiment, the preset compliance rules include a sensitivity index greater than or equal to a first preset threshold, a specificity index greater than or equal to a second preset threshold, and a cyclic threshold standard deviation less than or equal to a third preset threshold.

[0042] Secondly, this application also provides a device for evaluating the performance of MST primers in aquatic environments, comprising:

[0043] The data acquisition module is used to acquire water environment samples; the water environment samples include target pollutant samples and non-target microorganism samples.

[0044] The DNA extraction module is used to extract DNA from water environment samples and to standardize the concentration of the water environment samples to obtain positive and negative sample libraries.

[0045] The amplification curve and melting curve modules are used to perform amplification reactions with the target primers and the positive and negative sample libraries, respectively, to obtain amplification curve data and melting curve data. The amplification curve data includes positive amplification curves and negative amplification curves; the melting curve data includes positive melting curves and negative melting curves.

[0046] The index calculation module is used to obtain the sensitivity and specificity indices corresponding to the target primers based on melting curve data and amplification curve data.

[0047] The primer performance evaluation module is used to obtain primer performance results based on sensitivity indicators, specificity indicators, and cycle thresholds in amplification curve data; the primer performance results are used to characterize whether the primer performance meets the standards.

[0048] Thirdly, this application also provides a computer device, including a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the method described in the first aspect.

[0049] Fourthly, this application also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the method described in the first aspect.

[0050] The aforementioned methods, devices, equipment, and media for evaluating the performance of MST primers in aquatic environments ensure broad sample coverage by comprehensively collecting both target contaminant samples and non-target microbial samples, thus better simulating the actual aquatic environment. DNA concentration standardization eliminates interference from concentration differences between samples on PCR amplification, improving data accuracy. By combining key parameters such as melting curve melting temperature and amplification curve cycling threshold, a comprehensive evaluation is conducted from multiple dimensions, including sensitivity, specificity, and experimental repeatability. This addresses the problems of narrow sample coverage, inaccurate data, and incomplete indicators in traditional evaluation methods, enabling precise determination of primer performance and providing strong support for the reliability of MST detection in aquatic environments. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments or related technologies of this application, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 This is a flowchart illustrating a method for evaluating the performance of MST primers in aquatic environments, as described in one embodiment.

[0053] Figure 2 This is a schematic diagram of the structure of an MST primer performance evaluation device for the water environment in one embodiment;

[0054] Figure 3 This is a schematic diagram of a computer device in one embodiment. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0056] In one embodiment, such as Figure 1 As shown, a method for evaluating the performance of MST primers in aquatic environments is provided. This embodiment illustrates the application of this method to a primer performance evaluation terminal (hereinafter referred to as the terminal). It is understood that this method can also be applied to a server, or to a system including both a terminal and a server, and can be implemented through interaction between the terminal and the server. In this embodiment, the method includes the following steps:

[0057] S1, Obtain water environment samples.

[0058] The water environment samples include target contaminant samples and non-target microbial samples. Target contaminant samples include fecal samples from humans and various livestock. Non-target microbial samples include background flora. For example, in water bodies affected by human activities, the primer performance evaluation terminal uses a specific sampling device to collect fresh human and animal fecal samples as target contaminant samples. In the same water body, the primer performance evaluation terminal uses a specific sampling device to collect surface water samples away from the pollution source, filters and enriches suspended microorganisms, and extracts DNA as background flora, i.e., non-target microbial samples. The specific sampling device includes, but is not limited to, water sample collectors and filtration devices.

[0059] S2, DNA was extracted from the water environment samples, and the water environment samples were subjected to concentration standardization processing to obtain positive sample libraries and negative sample libraries.

[0060] Specifically, the primer performance evaluation terminal isolates microbial DNA from aquatic environmental samples and performs concentration standardization on the extracted DNA. The purpose of concentration standardization is to ensure that the DNA concentration of different samples is consistent, thereby eliminating experimental bias caused by concentration differences. Through standardization, positive and negative sample libraries are obtained. The positive sample library contains DNA from the target contaminated samples, while the negative sample library contains DNA from non-target microbial samples.

[0061] S3, using the target primers to perform amplification reactions with the positive and negative sample libraries respectively, to obtain amplification curve data and melting curve data.

[0062] The amplification curve data includes positive and negative amplification curves; the melting curve data includes positive and negative melting curves. Specifically, the primer performance evaluation terminal mixes the target primers with positive and negative sample libraries respectively, and performs amplification in a real-time PCR (Polymerase Chain Reaction) instrument. By monitoring the changes in fluorescence signals during the reaction process, positive and negative amplification curves are generated, i.e., amplification curve data, which reflect the change in fluorescence intensity with the number of cycles. After amplification, the amplification products are melted by heating, and the change in fluorescence signal with temperature is recorded to obtain positive and negative melting curves. The peak value of the melting curve can be used to reflect product specificity.

[0063] S4. Based on melting curve data and amplification curve data, the sensitivity and specificity indicators corresponding to the target primers are obtained.

[0064] Specifically, the primer performance evaluation terminal calculates sensitivity and specificity indicators based on melting curve and amplification curve data. Sensitivity indicators are derived by analyzing positive amplification curves, counting the proportion of all positive samples that can be successfully detected; sensitivity indicators reflect the primers' ability to detect target microorganisms. Specificity indicators are derived by analyzing negative amplification curves, counting the proportion of negative samples that do not show non-specific amplification; specificity indicators reflect the primers' ability to distinguish between target and non-target microorganisms.

[0065] S5. Primer performance results are obtained based on sensitivity indicators, specificity indicators, and cycle thresholds in amplification curve data.

[0066] Primer performance results are used to characterize whether primer performance meets the standards. Specifically, the primer performance evaluation terminal combines sensitivity indicators, specificity indicators, and the cycle threshold in the amplification curve data to comprehensively determine the primer performance results. The cycle threshold is the number of cycles at which the fluorescence signal reaches a preset threshold, reflecting the initial concentration of the target sequence and the amplification efficiency. By comparing the three indicators with preset standards, it is determined whether the primers meet the application requirements of the aquatic environment MST. The primer performance results directly indicate whether the primers meet the standards.

[0067] The above-mentioned method for evaluating the performance of water environment MST primers broadens the sample coverage by collecting target pollutant samples containing human and animal feces and non-target microbial samples containing environmental background flora, thus better reflecting the complex conditions of actual water environments. DNA extraction from water environment samples is standardized to avoid PCR amplification distortion caused by sample concentration differences, thereby improving data accuracy. Amplification curves and melting curves are obtained from positive and negative sample libraries. These data, along with cycle thresholds, are used to obtain sensitivity and specificity indicators, which are then comprehensively evaluated to improve the evaluation index system, avoid misjudgment of primer performance, and ensure the reliability of water environment MST detection.

[0068] To further illustrate the scheme of the embodiments of this application, the method for evaluating the performance of MST primers in the aquatic environment is described with reference to a specific example. In this embodiment, it is implemented based on the following method:

[0069] A primer performance evaluation system based on real-time PCR was established, and high-quality primers were screened using both sensitivity (>90%) and specificity (>90%) as dual indicators.

[0070] We innovatively introduced melting curve analysis technology and combined it with the standard deviation of Ct value (<0.5) to verify the amplification specificity.

[0071] Sample collection: Covering 7 host species in the Yellow River Basin (human, cattle, pig, chicken, dog, cat, and seagull).

[0072] DNA extraction: Tiangen fecal genomics kit was used, with a purity requirement of A260 / 280 = 1.8 ± 0.2.

[0073] Primer validation: PCR amplification was performed for 40 cycles, with a negative control (no template DNA) set up.

[0074] Data analysis: Sensitivity = number of true positives / total number of host samples; Specificity = number of true negatives / total number of non-host samples.

[0075] Media and reagents:

[0076] Primer synthesis: HPLC-purified primers (concentration 10 μM), that is, after primer synthesis, they are purified by HPLC and diluted to 10 μM.

[0077] Use Tiangen RealUniversal color fluorescence quantitative premixed reagent (containing SYBR Green I). Each reaction system contains 10 μL of 2×MasterMix, 0.4 μL of each primer, 2 μL of template DNA, and water to a final volume of 20 μL.

[0078] Reaction system: 2×RealUniversalMasterMix (containing ROXReferenceDye).

[0079] Key performance evaluation metrics:

[0080] Sensitivity is defined as the proportion of true positive host samples out of all host samples. The formula is: Sensitivity = (Number of true positive host samples detected / Total number of host samples detected) × 100%

[0081] For example, if a primer correctly identifies 95 out of 100 human samples, then the sensitivity is 95%.

[0082] Specificity: Defined as the proportion of negative host samples to the total expected negative host samples. Formula:

[0083] Specificity = (Number of negative host samples detected / Total number of expected negative host samples) × 100%

[0084] For example, if a primer misidentifies 2 out of 50 non-human samples, its specificity is 96%.

[0085] PCR instrument: An ABI 7300 real-time quantitative PCR instrument was used, and a three-step amplification program was set (pre-denaturation 95℃×10min, denaturation 95℃×15s, annealing 60℃×1min, extension 72℃×30s, for a total of 40 cycles).

[0086] Sequencing validation: The primer amplification products were sequenced using the Illumina MiSeq platform, and the specificity was validated by comparison with known microbial genome databases (such as NCBI's RefSeq).

[0087] Data recording: The melting curve was automatically generated using ABI 7300SDS software, and the mean and variance of the Ct value were calculated using Excel.

[0088] In an optional embodiment, DNA is extracted from aquatic environmental samples, and the samples are then subjected to concentration standardization to obtain a positive sample library and a negative sample library, including the following steps:

[0089] S11, DNA is extracted from the target contaminated sample to obtain a positive DNA template.

[0090] For example, the primer performance evaluation terminal performs DNA extraction on the target contaminated sample to obtain a positive DNA template. The target contaminated sample mainly comes from human and animal feces, which carry specific microbial markers and are the main targets of primer detection. The DNA extraction process involves separating the microbial DNA from the sample. Specific reagents and methods are required during the extraction process to ensure the integrity and purity of the DNA.

[0091] S12, DNA extraction is performed on non-target microbial samples to obtain negative DNA templates.

[0092] Specifically, the primer performance evaluation terminal performs DNA extraction on non-target microbial samples to obtain negative DNA templates. These non-target microbial samples mainly originate from environmental background microbiota, which are widely present in the aquatic environment. This background microbiota contrasts with the target contaminated samples, allowing for the assessment of primer specificity. The DNA extraction process involves separating the microbial DNA from the sample. Specific reagents and methods are required during extraction to ensure the integrity and purity of the DNA.

[0093] S13, the concentrations of positive and negative DNA templates are standardized to obtain positive and negative sample libraries with uniform concentrations.

[0094] Specifically, the primer performance evaluation terminal performs concentration standardization on the extracted positive and negative DNA templates. Concentration standardization ensures that the DNA concentration of different samples is consistent, eliminating experimental bias caused by concentration differences. Through standardization, a positive sample library and a negative sample library with uniform concentration are obtained. The positive sample library contains DNA from the target contaminated samples, while the negative sample library contains DNA from non-target microbial samples.

[0095] In an optional embodiment, amplification curve data and melting curve data are obtained based on a positive sample library and a negative sample library, including the following steps:

[0096] S21, based on the preset real-time PCR reaction system, the target primers are mixed with the positive sample library and the negative sample library respectively to obtain positive mixture and negative mixture.

[0097] Specifically, the primer performance evaluation terminal performs mixing operations according to a pre-set quantitative PCR reaction system. The quantitative PCR reaction system includes DNA polymerase (an enzyme that catalyzes DNA chain synthesis), dNTPs (Deoxynucleotide Triphosphates, raw materials for DNA synthesis), buffer (to maintain pH stability of the reaction system), fluorescent dye (to generate a fluorescent signal after binding to double-stranded DNA), and target primers (oligonucleotide chains that specifically recognize and bind to the target DNA sequence). The proportions of each component are optimized to ensure amplification efficiency. The terminal mixes the target primers with positive and negative sample libraries respectively to prepare positive and negative mixtures.

[0098] S22, the positive and negative mixtures are amplified separately to obtain the positive and negative amplification products corresponding to the target primers.

[0099] Specifically, the primer performance evaluation terminal places the positive and negative mixtures separately into a real-time PCR instrument for amplification. The amplification process follows a preset temperature cycling program, including three stages: denaturation (high temperature causes the DNA double strand to unwind into single strands), annealing (low temperature allows the primers to bind to complementary sequences of single-stranded DNA), and extension (DNA polymerase catalyzes the synthesis of daughter strands at intermediate temperatures). Multiple cycles achieve exponential amplification of the target DNA fragment. After amplification, the positive mixture produces a positive amplification product (the amplification product of the target microbial DNA), while the negative mixture either produces no target product if no non-specific reaction occurs, or produces a negative amplification product (the amplification product of non-target microbial DNA).

[0100] S23, based on positive and negative amplification products, amplification curve data and melting curve data are obtained.

[0101] For example, the primer performance evaluation terminal acquires curve data based on positive and negative amplification products. During amplification, the terminal monitors and records the fluorescence signal intensity in the reaction system in real time. As the number of cycles increases, the change in fluorescence intensity forms positive and negative amplification curves. The inflection point and slope of the curves reflect the amplification efficiency. After amplification, the amplification products are gradually defused through a stepwise procedure, and the fluorescence signal changes with temperature, resulting in positive and negative melting curves. A single peak indicates good specificity of the amplification product, while multiple peaks may indicate non-specific amplification.

[0102] In an optional embodiment, the amplification mixture is amplified to obtain amplification products and fluorescence intensity data, and the process further includes the following steps:

[0103] S31, the positive and negative amplification products are sequenced to obtain the sequencing results.

[0104] For example, the primer performance evaluation terminal sequences both positive and negative amplification products. Nucleic acid sequencing technology (such as Sanger sequencing, which terminates DNA chain extension with dideoxynucleotides and determines the base sequence via electrophoresis) is used to determine the base sequence of the amplification products, obtaining sequencing results containing specific base arrangement information. The sequencing process can verify whether the amplification product is a specific DNA fragment of the target microorganism. The sequencing results of positive amplification products should be consistent with the target sequence; if the sequencing results of negative amplification products do not match the target sequence, it indicates that the primers have good specificity.

[0105] S32, compare and verify the sequencing results with the preset microbial genome database to obtain the comparison and verification results.

[0106] The alignment verification results are used to characterize the specificity of the primers being tested. Specifically, the primer performance evaluation terminal compares the sequencing results with a pre-set microbial genome database. This database contains complete genome sequences of various known microorganisms, covering the genetic information of both the target microorganism and common non-target microorganisms. The terminal uses sequence alignment algorithms (such as BLAST, Basic Local Alignment Search Tool, which matches sequences by finding homologous regions) to analyze the degree of matching between the sequencing results and the sequences in the database. If the sequencing results of the positive amplification product highly match the target microorganism sequence, and the sequencing results of the negative amplification product have a low degree of matching with the target microorganism sequence, the alignment verification results indicate that the primers have good specificity.

[0107] In an optional embodiment, sensitivity and specificity indicators are obtained based on melting curve data and amplification curve data, including the following steps:

[0108] S41, extract the cycle threshold of all positive samples from the positive amplification curve to obtain the total amount of all host samples detected.

[0109] Specifically, the primer performance evaluation terminal extracts the cycle thresholds of all positive samples from the amplification curve data in the positive sample library. The cycle threshold refers to the number of PCR cycles at which the fluorescence signal first exceeds a set threshold, reflecting the initial concentration of the target DNA in the sample. By extracting the cycle thresholds of all positive samples, the total amount of all host samples detected can be obtained. The statistical results of the total amount of all detected host samples are used to reflect the primer's amplification efficiency and detection capability in positive samples.

[0110] S42, select the number of positive samples whose cycle threshold is less than the preset amplification threshold to obtain the number of true positive host samples detected.

[0111] For example, the primer performance evaluation terminal selects the number of positive samples with a cycling threshold lower than a preset amplification threshold to determine the amount of true positive host samples detected. The preset amplification threshold is a set cycling threshold standard used to distinguish between effective and ineffective amplification. Samples with a cycling threshold lower than this preset threshold are considered true positive samples, meaning that the target microorganism is indeed present and successfully amplified by the primers. The amount of true positive host samples detected reflects the primers' ability to accurately identify and amplify the target microorganism in actual detection.

[0112] S43, Based on the total number of all host samples detected and the total number of true positive host samples detected, the sensitivity index is calculated using the following formula:

[0113]

[0114] Among them, S sens P is a sensitivity indicator, where P represents the amount of true positive host sample detected. total This represents the total amount of all host samples detected.

[0115] Here, the number of true positive host samples detected refers to the number of positive samples whose cycle threshold is less than the preset amplification threshold, and the total number of all host samples detected refers to the sum of the cycle thresholds of all samples in the positive sample library. In the above formula for calculating the sensitivity index, S... sens The sensitivity index is indicated by the primer's efficiency in detecting the target microorganism, meaning it can more effectively identify and amplify the target microorganism's DNA sequence. P represents the amount of true positive host sample detected. total This represents the total amount of host samples to be detected. The sensitivity index calculated using this formula quantifies the accuracy of primers in detecting target microorganisms.

[0116] S44 extracts the cycle threshold of all negative samples from the negative amplification curve to obtain the total number of all expected negative host samples.

[0117] For example, the primer performance evaluation terminal extracts the cycle thresholds of all negative samples from the amplification curve data in the negative sample library to obtain the total number of all expected negative host samples. The samples in the negative sample library do not contain the target microorganism, and the cycle thresholds are used to reflect the amplification performance of the primers in non-target microorganism samples. After extracting the cycle thresholds of all negative samples, the total number of all expected negative host samples can be obtained. The statistical results of the total number of all expected negative host samples are used to reflect the amplification efficiency and specificity of the primers in negative samples.

[0118] S45, select the number of negative samples whose cycle threshold is greater than the preset amplification threshold to obtain the number of negative host samples detected.

[0119] For example, the primer performance evaluation terminal selects the number of negative samples with a cycling threshold greater than a preset amplification threshold to determine the amount of negative host samples detected. The preset amplification threshold is a set cycling threshold standard used to distinguish between effective and ineffective amplification. Samples with a cycling threshold greater than this preset threshold are considered as detected negative samples, i.e., the target microorganism is absent or the primer failed to amplify. The amount of negative host samples detected reflects the primer's ability to accurately distinguish non-target microorganisms in actual detection.

[0120] S46. Based on the total number of all expected negative host samples and the number of negative host samples detected, the specificity index is calculated using the following formula:

[0121]

[0122] Among them, S spec This is a specific indicator, where N is the number of negative host samples detected. total This represents the total number of all expected negative host samples.

[0123] The detected negative host sample quantity refers to the number of negative samples whose cycle threshold is greater than the preset amplification threshold, and the total expected negative host sample quantity refers to the sum of the cycle thresholds of all samples in the negative sample library. In the calculation formula of the above specificity index, S... spec The specificity index is a key indicator; a higher specificity index indicates that the primers are more efficient at distinguishing between target and non-target microorganisms, and can more effectively avoid false detections of non-target microorganisms. N represents the number of negative host samples detected. total This represents the total number of expected negative host samples. The specificity index calculated using this formula quantifies the accuracy of primers in distinguishing between target and non-target microorganisms.

[0124] In an optional embodiment, primer performance results are obtained based on sensitivity indicators, specificity indicators, and cycle thresholds in amplification curve data, including the following steps:

[0125] S51, repeat the experiment multiple times on the positive amplification curve to obtain multiple sets of cycle thresholds.

[0126] For example, the primer performance evaluation terminal performs multiple replicate experiments on the positive amplification curve. Each experiment uses the same positive sample, target primer, and reaction system, and performs quantitative real-time PCR amplification under the same conditions to reduce the impact of random errors on the results. Through multiple experiments, the terminal obtains multiple sets of cycle thresholds, each set containing the cycle thresholds of all positive samples in the experiment.

[0127] S52 calculates the average value of multiple sets of cyclic thresholds to obtain the average value set.

[0128] For example, the primer performance evaluation terminal calculates the average value of multiple sets of cycle thresholds obtained from repeated experiments, resulting in an average value set. Average value calculation is a commonly used method in statistics to reduce random fluctuations in experimental data and improve data stability and reliability. By calculating the average value of each set of cycle thresholds, the resulting average value set can more accurately reflect the average amplification efficiency and stability of the primers in multiple experiments.

[0129] S53, calculate the standard deviation of the cycle threshold using the following formula:

[0130]

[0131] Where, σ Ct The standard deviation of the cyclic threshold. Let μ be the average number of cycles when the detection threshold is reached in the i-th repeated experiment, μ be the overall mean of the set of averages, and n be the number of repeated experiments.

[0132] Specifically, the standard deviation of the cycle threshold reflects the degree of variation in the cycle threshold of a primer in multiple repeated experiments and is a key indicator for evaluating the stability of primer performance. The smaller the standard deviation, the more stable the amplification efficiency of the primer under different experimental conditions, and the more reliable the results.

[0133] In the formula for the standard deviation of the cyclic threshold above, σ Ct The standard deviation of the cycle threshold is denoted as . The smaller the standard deviation, the more stable the amplification efficiency of the primers under different experimental conditions, and the more reliable the results. Let Ct be the average of the number of cycles when the detection threshold is reached in the i-th repeated experiment, calculated by averaging the Ct values ​​from multiple repeated experiments on the same sample. μ is the overall mean of the set of averages, calculated by first averaging the Ct values ​​from each repeated experiment. Then these Take the average to obtain the population mean. n is the number of repeated experiments. The more repeated experiments there are, the more accurately the calculated standard deviation will reflect the repeatability of the experiment.

[0134] S54. Based on the sensitivity index, specificity index, and standard deviation of the cycle threshold, combined with the preset achievement rules, the primer performance results are obtained.

[0135] For example, the primer performance evaluation terminal derives primer performance results based on sensitivity indicators, specificity indicators, and cycle threshold standard deviation, combined with preset compliance rules. Sensitivity indicators reflect the primer's ability to detect target microorganisms, specificity indicators reflect the primer's ability to distinguish between target and non-target microorganisms, and cycle threshold standard deviation reflects the primer's stability and reliability in repeated experiments. The preset compliance rules are the standards for evaluating whether primer performance meets the requirements; only when primer performance simultaneously meets all these rules can it be considered to have met the compliance standards.

[0136] In an optional embodiment, the preset compliance rules include a sensitivity index greater than or equal to a first preset threshold, a specificity index greater than or equal to a second preset threshold, and a cyclic threshold standard deviation less than or equal to a third preset threshold.

[0137] Specifically, in practice, the primer performance evaluation terminal compares the calculated sensitivity, specificity, and cycle threshold standard deviation with preset compliance rules one by one. If the sensitivity is below the first preset threshold, it indicates that the primer may miss some targets when detecting the target microorganism; if the specificity is below the second preset threshold, it indicates that the primer may falsely detect non-target microorganisms; if the cycle threshold standard deviation is above the third preset threshold, it indicates that the primer's performance is not stable enough in repeated experiments, which may lead to unreliable results. Only when all three indicators meet the preset compliance rules will the primer performance evaluation terminal determine that the primer performance is up to standard; otherwise, further optimization and improvement of the primer are required.

[0138] The aforementioned method for evaluating the performance of MST primers in aquatic environments broadens the sample coverage by collecting target pollutant samples from human and animal feces, as well as non-target microbial samples from environmental background flora, thus better reflecting the complexities of actual aquatic environments. The DNA template is standardized to eliminate PCR amplification distortion caused by differences in sample concentration, improving data accuracy. Furthermore, by combining amplification curve data, melting curve data, and cycle thresholds, sensitivity and specificity indicators, along with the standard deviation of the cycle threshold, are introduced to construct a comprehensive evaluation system. This avoids misjudgments of primer performance due to a single indicator, ultimately ensuring the accuracy and reliability of primer performance evaluation and providing support for the effective application of MST detection technology in aquatic environments.

[0139] It should be understood that although the steps in the flowcharts of the embodiments described above are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the embodiments described above may include multiple steps or multiple stages. These steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the steps or stages of other steps.

[0140] Based on the same inventive concept, this application also provides an apparatus for evaluating the performance of MST primers in the water environment to implement the aforementioned method. The solution provided by this apparatus is similar to the solution described in the above method. Therefore, the specific limitations of one or more embodiments of the apparatus for evaluating the performance of MST primers in the water environment provided below can be found in the limitations of the method for evaluating the performance of MST primers in the water environment described above, and will not be repeated here.

[0141] In one exemplary embodiment, such as Figure 2 As shown, an aquatic environment MST primer performance evaluation device 200 is provided, comprising:

[0142] Data acquisition module 201 is used to acquire water environment samples; the water environment samples include target pollutant samples and non-target microbial samples;

[0143] DNA extraction module 202 is used to extract DNA from water environment samples and perform concentration standardization processing on the water environment samples to obtain positive sample library and negative sample library;

[0144] The amplification curve and melting curve module 203 is used to perform amplification reactions with the target primers and the positive sample library and the negative sample library, respectively, to obtain amplification curve data and melting curve data; the amplification curve data includes positive amplification curve and negative amplification curve; the melting curve data includes positive melting curve and negative melting curve;

[0145] The index calculation module 204 is used to obtain the sensitivity index and specificity index of the target primer based on melting curve data and amplification curve data.

[0146] The primer performance evaluation module 205 is used to obtain primer performance results based on sensitivity indicators, specificity indicators, and cycle thresholds in amplification curve data; the primer performance results are used to characterize whether the primer performance meets the standards.

[0147] Furthermore, the DNA extraction module 202 is also used for:

[0148] DNA was extracted from the target contaminated sample to obtain a positive DNA template.

[0149] DNA was extracted from non-target microbial samples to obtain negative DNA templates;

[0150] The concentrations of positive and negative DNA templates were standardized to obtain positive and negative sample libraries with uniform concentrations.

[0151] Furthermore, the amplification curve and melting curve module 203 includes:

[0152] The amplification mixing subunit is used to mix the target primers with the positive sample library and the negative sample library respectively based on the preset real-time PCR reaction system to obtain positive mixture and negative mixture;

[0153] The amplification product subunit is used to amplify the positive and negative mixtures separately to obtain the positive and negative amplification products corresponding to the target primers.

[0154] The curve data subunit is used to obtain amplification curve data and melting curve data based on positive and negative amplification products.

[0155] Furthermore, the amplified product subunit is also used for:

[0156] The positive and negative amplification products were sequenced to obtain the sequencing results.

[0157] The sequencing results were compared and verified with a pre-set microbial genome database to obtain the verification results; the verification results were used to characterize the specific performance of the primers to be tested.

[0158] Furthermore, the indicator calculation module 204 is also used for:

[0159] The cycle thresholds for all positive samples are extracted from the positive amplification curves to obtain the total amount of host samples detected.

[0160] Select the number of positive samples whose cycle threshold is less than the preset amplification threshold to obtain the amount of true positive host samples detected.

[0161] Based on the total number of host samples detected and the total number of true positive host samples detected, the sensitivity index is calculated using the following formula:

[0162]

[0163] Among them, S sens P is a sensitivity indicator, where P represents the amount of true positive host sample detected. total The total amount of all host samples detected;

[0164] Extract the cycle threshold of all negative samples from the negative amplification curve to obtain the total number of all expected negative host samples;

[0165] Select the number of negative samples whose cycle threshold is greater than the preset amplification threshold to obtain the number of negative host samples detected.

[0166] Based on the total number of all expected negative host samples and the number of negative host samples detected, the specificity index is calculated using the following formula:

[0167]

[0168] Among them, S spec This is a specific indicator, where N is the number of negative host samples detected. total This represents the total number of all expected negative host samples.

[0169] Furthermore, the primer performance evaluation module 205 is also used for:

[0170] The positive amplification curve was repeatedly tested to obtain multiple sets of cyclic thresholds.

[0171] The average value of multiple sets of cyclic thresholds is calculated to obtain the average value set;

[0172] Calculate the standard deviation of the cycle threshold using the following formula:

[0173]

[0174] Where, σ Ct The standard deviation of the cyclic threshold. Let μ be the average number of cycles when the detection threshold is reached in the i-th repeated experiment, μ be the overall mean of the set of averages, and n be the number of repeated experiments.

[0175] Based on sensitivity indicators, specificity indicators, and cycle threshold standard deviation, combined with preset achievement rules, primer performance results are obtained.

[0176] Furthermore, the preset compliance rules include a sensitivity index greater than or equal to a first preset threshold, a specificity index greater than or equal to a second preset threshold, and a cyclic threshold standard deviation less than or equal to a third preset threshold.

[0177] In one embodiment, such as Figure 3 A computer device 300 is provided, comprising:

[0178] At least one processor 301, and at least one memory 302 communicatively connected to said processor 301; said memory stores application code executable by said processor, said application code being executed by said processor to enable said processor to perform the steps of the water environment MST primer performance evaluation method as described above;

[0179] The computer device may also include: sensor 303;

[0180] The processor 301, memory 301, and sensor 303 can be connected via bus 304 or other means. The figure shows an example of connection via bus 304. Figure 3 The character is represented by a single thick line, but this does not mean that there is only one bus or a type of bus.

[0181] In one embodiment, a computer-readable storage medium is provided having a computer program stored thereon, which, when executed by a processor, implements the steps in the above method embodiments.

[0182] For the device embodiments, since they basically correspond to the method embodiments, the relevant parts can be referred to in the description of the method embodiments. The device embodiments described above are merely illustrative. The components described as separate parts may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this disclosure according to actual needs. Those skilled in the art can understand and implement this without creative effort.

[0183] The above-described embodiments are merely illustrative of several implementation methods of the embodiments of this application, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the embodiments of this application, and these modifications and improvements all fall within the protection scope of the embodiments of this application.

Claims

1. A method for evaluating the performance of MST primers in aquatic environments, characterized in that, The method includes: Obtain water environment samples; the water environment samples include target pollutant samples and non-target microorganism samples; DNA was extracted from the water environment samples, and the water environment samples were subjected to concentration standardization to obtain a positive sample library and a negative sample library; The target primers were used to perform amplification reactions with the positive sample library and the negative sample library, respectively, to obtain amplification curve data and melting curve data; the amplification curve data included positive amplification curves and negative amplification curves; the melting curve data included positive melting curves and negative melting curves. Based on the melting curve data and the amplification curve data, the sensitivity index and specificity index corresponding to the target primer are obtained; Based on the sensitivity index, specificity index, and the cycle threshold in the amplification curve data, primer performance results are obtained; these results are used to characterize whether the primer performance meets the standards.

2. The method according to claim 1, characterized in that, The process of extracting DNA from the water environment samples and standardizing the concentration of the water environment samples to obtain a positive sample library and a negative sample library includes: DNA was extracted from the target contaminated sample to obtain a positive DNA template; DNA was extracted from the non-target microbial sample to obtain a negative DNA template; The concentrations of the positive and negative DNA templates were standardized to obtain a positive and negative sample library with uniform concentrations.

3. The method according to claim 1, characterized in that, The step involves using the target primers to perform amplification reactions with the positive sample library and the negative sample library, respectively, to obtain amplification curve data and melting curve data, including: Based on a pre-defined quantitative PCR reaction system, the target primers are mixed with the positive sample library and the negative sample library respectively to obtain a positive mixture and a negative mixture. The positive and negative mixtures were amplified separately to obtain the positive and negative amplification products corresponding to the target primers. Based on the positive and negative amplification products, amplification curve data and melting curve data are obtained.

4. The method according to claim 3, characterized in that, After performing amplification processing on the positive and negative mixtures respectively to obtain the positive and negative amplification products corresponding to the target primers, the method further includes: Sequencing of the positive and negative amplification products yielded sequencing results. The sequencing results are compared and verified with a preset microbial genome database to obtain the comparison and verification results; the comparison and verification results are used to characterize the specific performance of the target primer.

5. The method according to claim 1, characterized in that, The process of obtaining sensitivity and specificity indicators corresponding to the target primer based on the melting curve data and the amplification curve data includes: The cycle thresholds for all positive samples are extracted from the positive amplification curves to obtain the total amount of all host samples detected. Select the number of positive samples whose cycle threshold is less than the preset amplification threshold to obtain the amount of true positive host samples detected. Based on the total number of host samples detected and the total number of true positive host samples detected, the sensitivity index is calculated using the following formula: Among them, S sens P is a sensitivity indicator, where P represents the amount of true positive host sample detected. total The total amount of all host samples detected; The cycle thresholds for all negative samples are extracted from the negative amplification curves to obtain the total number of all expected negative host samples; Select the number of negative samples whose cycle threshold is greater than the preset amplification threshold to obtain the number of negative host samples detected; Based on the total number of all expected negative host samples and the number of negative host samples detected, the specificity index is calculated using the following formula: Among them, S spec This is a specific indicator, where N is the number of negative host samples detected. total This represents the total number of all expected negative host samples.

6. The method according to claim 1, characterized in that, The primer performance results are obtained based on the sensitivity index, specificity index, and cycle threshold in the amplification curve data, including: The positive amplification curve was subjected to repeated experiments to obtain multiple sets of cyclic thresholds; The average value of the multiple sets of cyclic thresholds is calculated to obtain an average value set; Calculate the standard deviation of the cycle threshold using the following formula: Where, σ Ct The standard deviation of the cyclic threshold. Let μ be the average number of cycles when the detection threshold is reached in the i-th repeated experiment, μ be the overall mean of the set of averages, and n be the number of repeated experiments. Based on the sensitivity index, specificity index, and standard deviation of the cycle threshold, combined with the preset achievement rules, the primer performance results are obtained.

7. The method according to claim 6, characterized in that, The preset compliance rules include the sensitivity index being greater than or equal to a first preset threshold, the specificity index being greater than or equal to a second preset threshold, and the standard deviation of the cyclic threshold being less than or equal to a third preset threshold.

8. A device for evaluating the performance of MST primers in aquatic environments, characterized in that, The device includes: The data acquisition module is used to acquire water environment samples; the water environment samples include target pollutant samples and non-target microorganism samples. The DNA extraction module is used to extract DNA from the water environment sample and to perform concentration standardization processing on the water environment sample to obtain a positive sample library and a negative sample library. The amplification curve and melting curve module is used to perform amplification reactions with the positive sample library and the negative sample library using target primers, respectively, to obtain amplification curve data and melting curve data; the amplification curve data includes positive amplification curves and negative amplification curves; the melting curve data includes positive melting curves and negative melting curves; The index calculation module is used to obtain the sensitivity index and specificity index corresponding to the target primer based on the melting curve data and the amplification curve data. The primer performance evaluation module is used to obtain primer performance results based on the sensitivity index, specificity index, and the cycle threshold in the amplification curve data; the primer performance results are used to characterize whether the primer performance meets the standard.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that, When the processor executes the computer program, it implements the method of any one of claims 1 to 7.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the method of any one of claims 1 to 7.

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