A qPCR method for detecting residual host cell DNA

Abstract

The present invention belongs to the field of biotechnology and molecular diagnosis, and in particular, relates to a qPCR method for detecting residual host cell DNA. Specifically, a specific primer and probe combination for qPCR reaction for detecting residual Escherichia coli DNA in a sample is provided, wherein the specific primer and probe combination comprises: a forward primer sequence as shown in SEQ ID NO.1; a reverse primer sequence as shown in SEQ ID NO.2; and a probe sequence as shown in SEQ ID NO.3. The method of the present invention has high specificity, accuracy, and precision, and has passed a series of verifications, including range verification, quantitative limit verification, repeatability verification, and intermediate precision verification, to ensure its effectiveness and reliability in practical applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and molecular diagnosis, and particularly relates to a qPCR method for detecting host cell DNA residues. Background Art

[0002] In the field of biopharmaceuticals, Escherichia coli (E. coli) is widely used as a host cell to produce plasmid products due to its rapid growth and easy operation. As a widely studied and understood organism, the advantages of E. coli are its rapid proliferation ability, high adaptability to the production environment, and compatibility with biomanufacturing processes. These characteristics make E. coli an ideal host cell for the production of various vaccines, therapeutic proteins, and plasmid vectors for gene therapy. However, as a host cell, E. coli may cause its DNA to remain in the final product during the production process. This residue may not only reduce the purity and quality of the product, but also cause a series of biosafety issues, such as potential immunogenicity and carcinogenicity. The World Health Organization (WHO) stipulates that the acceptable amount of residual host cell DNA (HCD) is no more than 10 ng per dose. Therefore, strictly controlling and accurately measuring the level of host cell DNA in plasmid products, especially the residual DNA of E. coli, has become a vital task to ensure the quality and safety of biological products.

[0003] To address this challenge, the scientific and industrial communities have developed a variety of detection methods. Although traditional methods, such as gel electrophoresis and Southern blot analysis, are effective for detecting DNA residues, these methods are often time-consuming and insensitive, and are not suitable for rapid detection and quantification of trace DNA. Among the many detection methods, real-time quantitative polymerase chain reaction (qPCR) technology has become the preferred method due to its excellent sensitivity, accuracy, rapidity, and time efficiency [(DehuaLi et al., 2021)]. qPCR technology can accurately quantify trace DNA in a short time, which is particularly important for quality control. However, despite the obvious advantages of qPCR technology, existing qPCR methods still have certain limitations in dealing with complex sample matrices and ensuring detection specificity. In particular, when dealing with biological product samples containing multiple biological components and potential interferents, traditional qPCR methods may encounter problems such as nonspecific amplification, signal interference, and detection limitations. In addition, some existing qPCR technologies require complex sample pretreatment steps, which not only increases the complexity of the operation, but may also affect the final detection efficiency and accuracy. Therefore, developing an improved qPCR method that can provide high sensitivity and maintain high specificity while simplifying sample processing steps is crucial to improving the safety and quality control standards of biological products.

[0004] In response to these problems, the present invention proposes an improved qPCR method specifically for the detection of E.coli DNA residues in plasmid products. The qPCR method of the present invention significantly improves the sensitivity of detecting residual DNA by optimizing the design of primers and probes. Through this optimization, accurate detection results can be provided even in the case of extremely low levels of residual DNA. In addition, the specially designed primer and probe combination ensures a high degree of specificity for E.coli DNA, thereby reducing the possibility of nonspecific amplification. This is particularly important for preventing erroneous results, especially in complex sample matrices. In addition, the method of the present invention simplifies the sample processing steps, making the entire detection process faster and easier. This method is not only applicable to a variety of different types of plasmid product samples, but also can accurately detect residual DNA in complex sample matrices, thereby increasing the adaptability and practicality of the method. In summary, the qPCR method of the present invention provides an efficient, accurate and user-friendly solution for detecting the residues of E.coli DNA in plasmid products, which is significantly better than the prior art. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present application is to provide a qPCR method for detecting residual host cell DNA.

[0006] In one aspect, the present invention provides a specific primer and probe combination for qPCR reaction for detecting residual Escherichia coli DNA in a sample, wherein the specific primer and probe combination comprises:

[0007] The forward primer sequence is 5'-TGCTGGAAGGTAATCAAC-3', as shown in SEQ ID NO.1;

[0008] The reverse primer sequence is 5'-GGGATTTCAAACGATCCTC-3', as shown in SEQ ID NO.2;

[0009] The probe sequence is 5'-FAM-TCATTCTCACCTCGGATCGCT-BHQ1-3', as shown in SEQ ID NO.3.

[0010] In some embodiments, the E. coli DNA in the sample is the E. coli dnaA gene, and the dnaA gene sequence is shown in SEQ ID NO.4:

[0011]

[0012] In some embodiments, the amplification product size of the specific primer and probe combination is 84 bp.

[0013] In some embodiments, the sample is selected from a vaccine, a protein, a nucleic acid fragment, a plasmid, or a vitamin produced by E. coli.

[0014] On the other hand, the present invention provides a qPCR reaction kit for detecting residual Escherichia coli DNA in a plasmid product, wherein the kit comprises the aforementioned specific primer and probe combination.

[0015] In some embodiments, the kit further comprises an E. coli DNA standard. In some embodiments, the kit further comprises a qPCR reaction solution. In some embodiments, the kit further comprises an E. coli DNA standard and a qPCR reaction solution. In some embodiments, the qPCR reaction solution comprises: a dNTP mixture, an endonuclease RNase H, a hot start PCR enzyme, Mg 2+ solution and nuclease-free water.

[0016] In some embodiments, the qPCR reaction solution is Premix Ex Taq reagent, which is a commercial reagent of Takara, the full name is Premix Ex Taq (Probe qPCR), the product number is RR390A, and contains TaKaRa Ex Taq HS, dNTP mixture, Mg 2+ , Tli RNaseH, etc.

[0017] In some embodiments, when the purity of the purified and extracted E. coli DNA standard is tested, the test result reaches or exceeds 90%, preferably reaches or exceeds 95%, and more preferably is about 100%.

[0018] In some embodiments, the Escherichia coli DNA in the sample of the kit is the Escherichia coli dnaA gene, and the dnaA gene sequence is shown as SEQ ID NO.4.

[0019] In some embodiments, the kit has a minimum quantitative limit of 0.03 pg / μL for E. coli DNA in a plasmid product. In some embodiments, the kit has a detection range of 0.03 pg / μL to 300 pg / μL for E. coli DNA in a plasmid product.

[0020] On the other hand, the present invention provides a qPCR reaction method for detecting residual Escherichia coli DNA in a plasmid product, the method comprising the steps of using the aforementioned specific primer and probe combination or the aforementioned kit.

[0021] In some embodiments, the method comprises the following steps:

[0022] a) preparing a series of standard solutions of E. coli DNA;

[0023] b) preparing sample solution;

[0024] c) Prepare spiked sample solution;

[0025] d) preparing a qPCR mixed solution, which includes the steps of mixing the components of the aforementioned forward primer sequence, reverse primer sequence and probe sequence with Premix Ex Taq and nuclease-free water;

[0026] e) Add samples and perform qPCR detection;

[0027] f) Calculate and analyze the results.

[0028] In some embodiments, the method comprises the following steps:

[0029] a) preparing a series of standard solutions of E. coli DNA;

[0030] Take 10 μL of E. coli DNA standard (27.5 ng / μL), add 81.7 μL of NFW and mix well, mark it as ST0, and then dilute it in a 1:9 volume ratio to prepare standard solutions of subsequent concentrations, named ST1-ST5 respectively;

[0031] b) preparing sample solution;

[0032] Vortex the sample to mix well, dilute the sample to be tested to an appropriate concentration with NFW, and set aside;

[0033] c) Prepare spiked sample solution;

[0034] Take 100 μL of diluted sample, add 20 μL of ST2, and mix well;

[0035] d) preparing a qPCR mixed solution, which includes the step of mixing the components of the aforementioned forward primer sequence, reverse primer sequence and probe sequence with a qPCR reaction solution;

[0036] e) Add samples and perform qPCR detection;

[0037] f) Calculate and analyze the results.

[0038] Premix Ex Taq reagent is a commercial reagent from Takara, the full name is Premix Ex Taq (Probe qPCR), the product number is RR390A, and it contains TaKaRa Ex Taq HS, dNTP mixture, Mg 2+ , Tli RNaseH, etc.

[0039] The beneficial technical effects of the present invention are as follows:

[0040] In one aspect, the present invention proposes an improved real-time fluorescent quantitative polymerase chain reaction (qPCR) method, which focuses on improving the accuracy and efficiency of detecting Escherichia coli (E. coli) DNA residues in plasmid products. This method makes key optimizations to the existing technology, especially in terms of improving specificity, sensitivity and simplifying sample processing, so as to solve the shortcomings of traditional methods in terms of specificity, sensitivity and sample processing complexity.

[0041] The present invention significantly improves the detection sensitivity and specificity of E. coli DNA by using specially designed primers and probes targeting the DNA sequence unique to E. coli. These primers and probes can specifically bind to E. coli DNA during the qPCR process, thereby effectively reducing nonspecific amplification and background noise, and ensuring the accuracy of the detection results.

[0042] qPCR Condition Optimization: Carefully optimize the PCR reaction conditions, including cycle number, annealing temperature and time, to ensure maximum detection efficiency and accuracy.

[0043] Sensitivity and specificity testing: The sensitivity of the method was tested by performing qPCR in E. coli DNA samples with different concentrations. At the same time, cross-reaction experiments were performed using non-E. coli DNA samples to verify the specificity of the method.

[0044] Repeatability and precision evaluation: The repeatability and precision of the qPCR method were evaluated by repeated experiments to ensure its stability across different batches and experimental conditions.

[0045] In one embodiment, the present invention uses a carefully designed primer and probe combination to detect E. coli DNA residues with high sensitivity and specificity for specific DNA sequences of E. coli. The specific combination of these dedicated primers and probes in qPCR effectively reduces non-target amplification and background noise, and improves the accuracy of the detection results.

[0046] In one embodiment, the present invention relates to a method for preparing a standard substance, which aims to provide a stable reference substance for qPCR experiments by accurately controlling the concentration and purity. The accuracy and reliability of the preparation process are ensured through strict concentration calibration and purity detection steps, thereby providing a high-quality standard substance for detecting the residual amount of E. coli DNA.

[0047] In one embodiment, the present invention describes a comparative experiment with existing kits to demonstrate the advantages of the present invention in terms of consistency and reliability. By comparing the %CV values ​​of different kits, the improvement of the present method in detection consistency is demonstrated, especially in terms of reducing operational complexity and increasing detection speed.

[0048] In one embodiment, the present invention verifies the effective detection range of the qPCR method by measuring the recovery rate at different concentrations to ensure that the method can accurately quantify different levels of E. coli DNA residues.

[0049] In one embodiment, the present invention evaluates the limit of quantitation of the qPCR method by measuring the %CV and recovery of very low concentration samples, demonstrating the high sensitivity of the method in detecting low levels of DNA.

[0050] In one embodiment, the present invention demonstrates a specificity verification process for a specific DNA, and the high specificity of the method is verified by analyzing the spike recovery rate and the signal of non-target DNA samples.

[0051] In one embodiment, the present invention demonstrates the high accuracy of the qPCR method at different concentrations by analyzing the recovery of spiked samples.

[0052] In one embodiment, the present invention verifies through repeatability validation experiments that the qPCR method has a low %CV value in multiple repeated experiments, indicating the high repeatability of the method.

[0053] In one embodiment, the present invention demonstrates that the qPCR method has good intermediate precision by evaluating the %CV value under different experimental conditions, indicating its stability under various conditions.

[0054] In one embodiment, the present invention evaluates the stability of the qPCR method on different instruments, and by comparing the %CV values, demonstrates the consistency and durability of the method in different experimental equipment.

[0055] In one embodiment, the present invention is carried out by establishing a standard curve and analyzing R 2 The linearity of the qPCR method was verified by the PCR product and the slope was confirmed, ensuring its accurate quantification capability over a wide concentration range.

[0056] In addition, the present invention also includes the optimization of qPCR reaction conditions, such as the adjustment of the number of cycles, annealing temperature and time, to ensure the maximization of detection efficiency and accuracy. The sensitivity test was performed on E. coli DNA samples of different concentrations, and the specificity was verified by cross-reaction experiments using non-E. coli samples. The evaluation of repeatability and precision was completed through multiple experiments to ensure the stability and reliability of the method under different batches and conditions. These embodiments jointly support the application of the present invention in qPCR technology and demonstrate significant advantages over the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Primer distribution map. DETAILED DESCRIPTION

[0058] I. Definition

[0059] Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

[0060] As used herein, the term "polymerase chain reaction" (PCR) refers to a laboratory technique that allows millions to billions of copies of a specific DNA sequence to be rapidly replicated in vitro through temperature cycling. This technique is a cornerstone of the field of molecular biology and is widely used in medical diagnosis, forensics, and biological research.

[0061] As used herein, the term "real-time quantitative PCR" (qPCR) refers to an advanced form of PCR that integrates the function of real-time monitoring of fluorescent signals to quantitatively detect DNA during PCR amplification. This method can provide not only qualitative results but also quantitative information about the amount of DNA starting, and is an indispensable tool in modern biotechnology and clinical diagnosis.

[0062] As used herein, the term "Escherichia coli" (E. coli) refers to a widely studied bacterial model organism that is commonly found in the intestines of humans and other warm-blooded animals. While many strains are harmless, some strains can cause severe disease. E. coli is favored for its wide range of applications in gene cloning, expression systems, and microbiology research.

[0063] As used in this article, the term "Nuclease Free Water" (NFW) refers to water that has been specially treated to remove any enzymes that may degrade nucleic acid samples, ensuring a safe solution environment in molecular biology experiments and avoiding possible degradation of DNA or RNA samples.

[0064] As used in this article, the term "Extraction Recovery Control" (ERC) refers to a quality control measure that is usually added to the sample during the extraction of DNA or RNA. It serves as an internal standard to help assess the sample loss or degradation that may have occurred throughout the extraction process and ensure the reliability of the results.

[0065] As used herein, the term "Negative Template Control" (NTC) is used in the PCR reaction to remove any DNA template from the reaction mixture. NTC is a key control used to monitor contaminants or false positives in the experiment to ensure the accuracy and credibility of the PCR results.

[0066] As used herein, the term "standard" refers to a substance with known properties (e.g., concentration, purity) used as a benchmark for comparison in an experiment. In qPCR experiments, standards are often used to create a standard curve for calibrating instruments and ensuring the accuracy of experimental results.

[0067] As used herein, the term "spiked sample" means that a certain amount of target compound is added to the sample, after which a certain concentration of the target compound can be detected. The target compound concentration minus the blank (the concentration of the target compound detected in the sample without spike) is the sample spike recovery rate.

[0068] As used herein, the term "recovery" represents the ratio between the amount of target analyte extracted and detected from a sample and the known amount actually added. It is an important indicator for evaluating the extraction efficiency and accuracy of quantitative analytical methods. Ideally, a recovery close to 100% indicates that the analytical method has a high accuracy.

[0069] As used in this article, the term "coefficient of variation" (%CV) refers to the ratio of the standard deviation to the sample mean, which is used to measure the repeatability or consistency of experimental results. A lower %CV value indicates that the difference between experimental data is small and the repeatability is good. It is an important parameter for evaluating the stability and reliability of experimental operations.

[0070] As used herein, the term "amplification efficiency" refers to the efficiency of the target DNA molecule to replicate in each cycle during the qPCR process. The ideal amplification efficiency is close to 100%, indicating that the amount of DNA doubles after each PCR cycle. The amplification efficiency is an important indicator for evaluating whether the qPCR reaction settings are optimized.

[0071] As used herein, the term "specificity" refers to the ability of primers and probes to bind and amplify only target DNA sequences in qPCR technology, but not to non-target sequences. High specificity ensures the purity of the amplified product, thereby improving the accuracy of the test results.

[0072] As used herein, the term "sensitivity" refers to the ability of a detection method to detect low-abundance target DNA. Highly sensitive qPCR methods are able to detect extremely small amounts of DNA, which is critical for early pathogen detection and low-level gene expression analysis.

[0073] As used herein, the term "stability" refers to the ability of primers, probes, and other reaction components to maintain their performance over multiple experiments or after long-term storage. Stability directly affects the repeatability and reliability of long-term experiments.

[0074] As used herein, the term "limit of detection (LOD)" is also called the detection limit, which refers to the corresponding amount of 3 times the instrument background signal generated by the matrix blank, or the average value of the background signal generated by the matrix blank plus 3 times the mean standard deviation. It is one of the important indicators of the sensitivity of the method (method detection limit MDL) and the instrument (instrument detection limit IDL). In the test kit, it refers to the minimum concentration or minimum amount of the substance to be tested that can be detected from the sample within a given reliability. The so-called detection refers to qualitative detection, that is, it is determined that the substance to be determined exists in the sample at a concentration higher than the blank.

[0075] As used herein, the term "limit of quantitation (LOQ)" refers to the lowest amount of an analyte in a sample that can be quantitatively determined with a certain degree of accuracy. The LOQ reflects whether the analytical method has sensitive quantitative detection capabilities.

[0076] II. Examples

[0077] The specific embodiments of the invention focus on an improved real-time fluorescence quantitative polymerase chain reaction (qPCR) method for detecting Escherichia coli (E. coli) DNA residues in plasmid products. The experiment is based on qPCR technology. By using specially designed probes and primers, the dnaA gene fragment unique to E. coli is used to achieve high sensitivity and specificity detection of E. coli DNA residues. The qPCR reaction solution carries a probe, which is a single-stranded oligonucleotide that is complementary to the target DNA. There is a reporter group and a quencher group at both ends of the probe. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group, and no fluorescent signal is detected; when PCR amplification is performed, the polymerase in the qPCR reaction solution will enzymatically degrade the probe, separate the reporter group from the quencher group, and thus detect the fluorescent signal of the reporter group in real time. Each time a DNA chain is synthesized, a probe is cut off and a unit of fluorescent signal is generated. The signal intensity is proportional to the probe on the DNA chain.

[0078] The experiment used extracted and purified E. coli DNA as a standard, and its mass concentration was calibrated by the A260 value. The selected dnaA gene fragment is a highly conserved protein at the chromosome replication start site in the E. coli genome. Using primer probes designed for the dnaA gene fragment, the residual amount of E. coli DNA in unknown samples was quantitatively determined by qPCR technology combined with the standard curve, and the size of the amplified product was 84bp.

[0079] The information of the instruments and equipment used in the examples is shown in Table 1 below:

[0080] Table 1 Main instrument and equipment information

[0081]

[0082] Reagent information is as follows Table 2:

[0083] Table 2 Reagent information

[0084]

[0085] Primer distribution and design see Figure 1 , wherein the forward primer F-dnaA is located at the 770th to 787th nucleotides of dnaA, the reverse primer R-dnaA is located at the 835th to 853rd nucleotides of dnaA, and the probe is located at the 791st to 811th nucleotides of dnaA.

[0086] The experimental consumables information is as follows Table 3:

[0087] Table 3 Consumables information

[0088]

[0089] The specific information of the E. coli DNA standard used in the experiment is as follows: E. coli DNA standard, concentration is 27.5 ng / μL, and the storage condition is -70±10℃.

[0090] The experimental steps include: preparation of standard solution and test sample, preparation of spiked sample (ERC), preparation of qPCR Mix, addition of sample and qPCR detection.

[0091] (1) Preparation of standard solution

[0092] Specifically, first prepare a 3ng / μL ST0 standard solution, take 10μL E.coli DNA standard (27.5ng / μL), add it to 81.7μL NFW and mix well, and mark it as ST0 (3ng / μL). The preparation scheme of the standard solution is as shown in Table 4:

[0093] Table 4 Standard solution preparation

[0094]

[0095] (2) Preparation of test samples

[0096] Then, dilute the sample to be tested to an appropriate concentration and use it as a test sample. After the sample to be tested is fully melted, vortex mix. Use NFW to dilute the sample to be tested to an appropriate concentration (the dilution factor can be adjusted according to the amount of E. coli DNA residue in the sample to be tested) and use it as a test sample.

[0097] (3) Preparation of spiked samples (ERC)

[0098] The preparation of the spiked sample (ERC) included taking 100 μL of the diluted test sample, adding 20 μL of ST2 (30 pg / μL), and mixing by pipetting. The theoretical spiked amount was 600 pg.

[0099] (4) Preparation of qPCR mixed solution

[0100] When preparing qPCR Mix, you need to prepare according to the number of reaction wells required + 2 in Table 5 below to determine the total amount to prevent insufficient solution. After preparation, invert the plate at least 15 times to mix well and dispense into a qPCR 96-well plate, 20µL / well.

[0101] Table 5 qPCR Mix preparation

[0102]

[0103] (5) Add sample

[0104] The sample addition process is as shown in Table 6 below. The standard solution, test sample, spiked sample ERC and NTC (NFW) are added to the 96-well plate containing qPCR Mix. Three replicate wells are set for each sample, and 10 µL is added to each well.

[0105] Table 6 Sample addition diagram

[0106]

[0107] (6) qPCR detection

[0108] qPCR detection uses Bio-Rad instruments and software. The specific operation and reaction condition parameters are as follows:

[0109] Reaction conditions: 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, 40 cycles.

[0110] Sample volume: 30 µL. This is the total volume of each reaction mixture.

[0111] Software Setup and Sample Preparation:

[0112] Software settings: Use Bio-Rad qPCR software for operation, set the plate type (Plate Type) to BRWhite, and select FAM as the fluorescent probe.

[0113] Standard sample settings: Set the sample type of the standard sample (Sample Type) to Standard, set the number of technical replicates (Technical Replicates) to 3, set the starting concentration (Starting Concentration) and the dilution factor (Dilution Factor).

[0114] Sample setup: For the samples to be tested and NTC (no template control) samples, set the corresponding sample type and number of repetitions.

[0115] Run the experiment:

[0116] After completing all settings, save the plate file and start the qPCR instrument to run the experiment.

[0117] (7) Calculation of results

[0118] The calculation formula of the residual amount of the test plasmid is:

[0119] E.coli DNA residual amount (mg / mL) = reaction well reading value (pg / μL) × test sample dilution factor / (1E+6)

[0120] The formula for calculating the spike recovery rate is:

[0121] ,

[0122] In addition, the data validity is judged as shown in Table 7:

[0123] Table 7 Data validity determination

[0124]

[0125] The specific experiments are shown in Examples 1-10.

[0126] Example 1 Preparation of Standards

[0127] In this example, E. coli DNA standards were prepared to ensure the accuracy and reliability of the real-time fluorescence quantitative polymerase chain reaction (qPCR) method in detecting E. coli DNA residues in plasmid products.

[0128] The specific experimental steps are as follows:

[0129] Preparation: Use TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver 3.0 to extract and purify E. coli DNA from E. coli culture medium, divide into low-absorption screw tubes, 15 μL / tube, and store at -70±10℃.

[0130] Calibration: Two experimenters each took 6 E. coli DNA standards and tested the concentration in a NanoDrop UV spectrophotometer. Each sample was tested twice in parallel and the average of the two test results was taken as the calibration value.

[0131] Data Analysis:

[0132] The mean value and coefficient of variation (%CV) of the concentration calibration were calculated according to the above formula, and the acceptance standard was that the concentration calibration result %CV ≤ 5%.

[0133] The purity of E. coli DNA standards was tested by AGE method, and the results of purity test were evaluated. The acceptance standard was purity (AGE) ≥ 90%.

[0134] The experimental results showed that the concentration of the E. coli DNA standard was 27.5 ng / μL and the %CV value was 2%, indicating that the measurement results were highly consistent and accurate. The AGE purity test showed only one genomic band, and the results showed that the purity reached 100%, meeting the preset acceptance criteria. These data show that the prepared E. coli DNA standard is of high quality and suitable for subsequent qPCR experiments.

[0135] Example 2 Kit Comparison

[0136] This example evaluates and compares the consistency and reliability of the qPCR method of the present invention and a commercial E. coli DNA residue detection kit in detecting plasmid samples.

[0137] The specific experimental steps are as follows:

[0138] The qPCR method of the present invention was used to detect the residual amount of E. coli DNA in a series of plasmid samples, and a commercial reference kit was used to detect the same samples, and the coefficient of variation (%CV) of the detection results obtained by the two methods was calculated to evaluate the consistency.

[0139] Data Analysis:

[0140] Based on the experimental data obtained, the detection results of each sample were analyzed and the %CV of the two methods were calculated.

[0141] Compare the test results of the two methods to determine whether the %CV is within the acceptable range (≤30%).

[0142] The experimental results are shown in Table 8 below.

[0143] Table 8 Comparison of qPCR method and reference kit for detecting residual E. coli DNA

[0144]

[0145] Note: %CV (coefficient of variation) is used to reflect the consistency of the results between the two methods.

[0146] Low coefficient of variation: For example, the %CV of samples 1 and 5 was 1%, indicating that the qPCR method had extremely high consistency with the commercial reference kit, indicating the reliability of the experimental method.

[0147] High coefficients of variation: %CVs are 12% and 23% for Sample 2 and Sample 3, respectively. These higher coefficients of variation may be due to differences in sensitivity, specificity, or other factors that affect assay performance.

[0148] Concentration variability: The %CV varies between different concentrations of E. coli DNA, and the %CVs for lower concentration samples (such as samples 4 and 9) are relatively high, indicating that the performance of the experimental method may vary at different DNA concentrations.

[0149] Assay sensitivity: The developed qPCR assay was able to detect very low E. coli DNA concentrations (0.9 pg / μL), although the %CV was 13%, indicating some room for optimization.

[0150] By analyzing the data in Table 8, it can be seen that the %CV values ​​of the detection results of all samples are between 1% and 23%. This shows that the qPCR method of the present invention and the commercial reference kit show good consistency in detecting plasmid samples, and both meet the acceptance criteria.

[0151] Example 3 Range Verification

[0152] In this example, the quantitative PCR (qPCR) method was used to verify the recovery rate of the diluted samples of the E. coli DNA standard within a certain concentration range to determine the accuracy of the qPCR method at different concentration levels.

[0153] The specific experimental steps are as follows:

[0154] Prepare a range of samples by diluting the E. coli DNA standard to seven different concentration levels, from 300pg / μL to 0.0003pg / μL. Take the E. coli DNA standard and dilute it with preparation buffer to 300pg / μL, 30pg / μL, 3pg / μL, 0.3pg / μL, 0.03pg / μL, 0.003pg / μL, 0.0003pg / μL, and perform three replicates for each concentration level.

[0155] Data Analysis:

[0156] Calculate the recovery of each range sample by comparing the DNA concentration measured by qPCR to the actual known DNA concentration. Analyze whether the recovery of the range samples meets the set acceptance criteria, which is between 70% and 130%.

[0157] The test data of samples with different concentrations are shown in Table 9 below.

[0158] Table 9 Data on the effect of sample concentration on recovery rate

[0159]

[0160] As shown in Table 9, the recoveries of samples Range-1 to Range-5 ranged from 96% to 121%, which met the acceptance criteria. However, the recoveries of Range-6 and Range-7 were 214% and 1316%, respectively, exceeding the acceptance criteria, indicating that at very low concentration levels (below 0.03 pg / μL), the accuracy of the current qPCR method is not as good as at high concentrations.

[0161] Example 4 Quantification Limit Verification

[0162] In this example, the limit of quantification (LOQ) of the qPCR method was verified, that is, the level at which the lowest concentration of E. coli DNA can be accurately detected.

[0163] The experimental steps are as follows:

[0164] Prepare 8 LOQ samples and LOQ spike samples, dilute to a concentration of 0.03 pg / μL, dilute the E. coli DNA standard with preparation buffer, prepare 8 LOQ samples (0.03 pg / μL) and 8 LOQ spike samples in parallel, and perform three replicates on each.

[0165] Data Analysis:

[0166] The detection value, %CV value and recovery rate of each LOQ sample were calculated based on the qPCR results.

[0167] The same analysis was performed on the LOQ spiked samples to determine the spike recoveries.

[0168] The test data of different samples are shown in Table 10 below.

[0169] Table 10 Test results of different samples

[0170]

[0171] From the test results in Table 10, it can be seen that the recovery rate of the LOQ samples ranged from 108% to 126%, which is between 70% and 130% of the acceptance standard, indicating that the qPCR method is accurate in detecting E. coli DNA within this concentration range. The %CV value is 6%, which is much lower than the acceptance standard of 30%, indicating that the test results have good consistency and repeatability.

[0172] The detection values ​​and recoveries of the spiked samples also provided important information. Except for the spiked recovery of LOQ-8 which was slightly lower than the minimum limit of the acceptance range (87% vs 90%), the spiked recoveries of the remaining samples were within the acceptance range, ranging from 90% to 120%.

[0173] The validated qPCR method was effective and reliable at a limit of quantification of 0.03 pg / μL, suitable for the precise detection of E. coli DNA residues.

[0174] Example 5 Specificity Verification

[0175] To ensure that the qPCR method can specifically detect E. coli DNA without cross-reaction with other similar biomolecules, this example further verifies its specificity.

[0176] The specific experimental steps are as follows:

[0177] Take the preparation buffer, E. coli RNA quantitative reference, and HEK293 DNA quantitative reference as specific samples and perform a test once.

[0178] Compare the Ct values ​​and spike recoveries of the formulation buffer. The acceptance criteria are as follows:

[0179] Preparation buffer, E. coli RNA quantitative reference, HEK293 DNA quantitative reference>ST5 Ct value or N / A.

[0180] The spike recovery rate is between 70% and 130%.

[0181] The sample Ct values ​​and spike recovery rates are shown in Table 11. Since E. coli RNA and HEK293 DNA are not the targets of this qPCR test, ideally there should be no Ct value (N / A) and the spike recovery rate should have no cross-reaction with E. coli DNA.

[0182] Table 11 Sample Ct values ​​and spike recovery rates

[0183]

[0184] As shown in Table 11, the Ct value of the preparation buffer was 35.24, the spike recovery rate was 123%, and the spike recovery rates of E. coli RNA and HEK293 DNA were 123% and 99%, respectively, both within the preset recovery range of 70% to 130%. This result shows that the qPCR method has good specificity and can specifically detect E. coli DNA without being affected by the preparation buffer, E. coli RNA or HEK293 DNA.

[0185] Example 6 Accuracy Verification

[0186] In this example, the performance of the detection method at different concentration levels was evaluated by the recovery rate of the spiked samples, and the accuracy of the qPCR detection method was verified.

[0187] The specific experimental steps are as follows:

[0188] Take sample 10 and dilute it 500 times with NFW, and mark it as Accuracy-S1; take sample 11 and dilute it 100 times with NFW, and mark it as Accuracy-S2. Prepare spiked accuracy samples at three concentration levels: high, medium, and low according to Table 12.

[0189] Table 12 Preparation of spiked sample concentration table

[0190]

[0191] Data Analysis:

[0192] Calculate the recoveries of high, medium and low concentration spiked samples respectively, compare the recoveries with the theoretical spiked amount, and determine whether they are within the acceptance standard range of 50% to 150%. The specific data are shown in Table 13 below.

[0193] Table 13 Accuracy of samples of different concentrations

[0194]

[0195] From the results in Table 13, we can see that the high, medium, and low spike recoveries of sample 10 are 119%, 120%, and 126%, respectively. The high and medium spike recoveries of sample 11 are 124% and 127%, and the low spike recovery is 64%, which is slightly lower than the expected range, but is between 50% and 150%, meeting the acceptance criteria. This shows that at these concentration levels, the qPCR method can accurately detect spiked E. coli DNA.

[0196] Example 7 Repeatability Verification

[0197] In this example, the repeatability of sample 10 and sample 11 was verified by qPCR technology, that is, the consistency of the results obtained by continuous measurement of the same sample under the same experimental conditions.

[0198] The specific experimental steps are as follows:

[0199] Six replicates of the test article and spiked samples of sample 10 and six replicates of sample 11 were prepared in parallel by a single experimenter. Each test article and spiked sample was subjected to a single qPCR assay.

[0200] Data Analysis:

[0201] The coefficient of variation (%CV) of the six test samples and six spiked samples was calculated to assess repeatability. The acceptance criteria for %CV was %CV ≤ 30%. The results are shown in Table 14 below.

[0202] Table 14 Sample repeatability

[0203]

[0204] As shown in Table 14, the residual E. coli DNA values ​​of sample 10 varied between 873.31 and 1016.54 pg / μL, with a %CV of 5%, showing very small variation, indicating that the test was highly repeatable. The spike recovery ranged from 87% to 149%, with a %CV of 19%, and most of the data points were within the acceptance criteria (70%-130%), except for one data point of 149%, which was slightly higher.

[0205] The residual E. coli DNA values ​​for sample 11 were between 224.08 and 269.87 pg / μL with a %CV of 6%, also indicating good repeatability. The spike recovery was between 101% and 121% with a %CV of 7%, and all data points were within the acceptance criteria.

[0206] All %CV values ​​were below the acceptance criteria of 30%, indicating good reproducibility of the qPCR assay for both samples.

[0207] Example 8 Intermediate Precision Verification

[0208] This example verifies the intermediate precision of the qPCR method, that is, the consistency between repeated experiments under different experimental conditions.

[0209] The specific experimental plan is as follows:

[0210] On different days, the same experimenter took samples that had been previously verified for repeatability, prepared 6 test samples and 6 spiked samples in parallel, and performed qPCR detection.

[0211] Data Analysis:

[0212] Calculate the %CV values ​​of the 6 test samples and spiked samples on different days, combine the data from the repeatability verification, and calculate the %CV values ​​of a total of 12 test samples and spiked samples. The acceptance criteria is %CV ≤ 30%, and the results are shown in Table 15 below.

[0213] Table 15 Intermediate precision verification data

[0214]

[0215] As shown in Table 15, for sample 10, the %CV of E. coli DNA residue was 5% in the repeatability experiment and 5% in the intermediate precision experiment, both showing very low variability, indicating that the experimental results were consistent at different time points. In the repeatability experiment, the %CV of the spike recovery was 19%, while in the intermediate precision experiment it was slightly higher at 27%, but still within the acceptance standard of 30%.

[0216] Sample 11 also showed good consistency with a %CV of 6% in the repeatability test and 7% in the intermediate precision test. The %CV of spike recovery in the repeatability test and intermediate precision test were 7% and 10%, respectively, which were also within the acceptable range.

[0217] The results showed that the qPCR method was able to produce consistent and reliable results with coefficients of variation within acceptable limits over intermediate time scales, which underscores the robustness and accuracy of this assay.

[0218] Example 9 Durability Verification

[0219] In order to ensure that the stability and reliability of the qPCR method are not affected by instrument replacement and long-term storage of the solution, this example compares the results under different conditions and evaluates the durability of the method. Instrument durability verification ensures that the method can provide reliable results on different instruments, while sample solution stability verification ensures that the sample can still provide consistent test results after storage.

[0220] Experimental protocol:

[0221] 1) Instrument robustness: Replicate previous validation experiments on different qPCR instruments.

[0222] 2) Solution stability: The previous repeatability verification samples were stored at 2 to 8 degrees Celsius for 2 hours before qPCR testing.

[0223] Data Analysis:

[0224] Calculate the %CV of the six test and spiked samples on the new instrument, as well as the %CV after a short storage period. Compare these %CV values ​​to the acceptance criteria, which are:

[0225] 1) The %CV of 6 replicates of the test product and the spiked sample is ≤30%.

[0226] 2) Repeatability: %CV≤30% for 12 samples of test and spiked samples.

[0227] The result data are shown in Tables 16 and 17 below:

[0228] Table 16 Instrument durability

[0229]

[0230] Table 17 Solution stability

[0231]

[0232] in conclusion:

[0233] 1) Instrument durability:

[0234] The %CV of E. coli DNA residue in sample 10 was 5% in the instrument durability test, which was consistent with the repeatability test (5%), indicating that the results were consistent across instruments. The %CV of spike recovery was 23% in the instrument durability test, which was an increase from the repeatability test (19%), but still below the acceptance standard of 30%.

[0235] The %CV of E. coli DNA residue in sample 11 was 7% in the instrument durability test, which was close to the repeatability test (6%), showing good consistency. The %CV of spike recovery was 4% in the instrument durability test, which was reduced compared to the repeatability test (7%), indicating stability on different instruments.

[0236] 2) Solution stability:

[0237] The %CV of residual E. coli DNA in sample 10 was 6% in the solution stability test, which is consistent with the repeatability test (5%), indicating that the sample is stable after short-term storage. The %CV of spike recovery increased significantly to 28% in the solution stability test, which may indicate that the stability of the sample was affected during storage.

[0238] The %CV of E. coli DNA residue in sample 11 was 8% in the solution stability test, slightly higher than the repeatability test (6%), but still within the acceptable range. The %CV of spike recovery was 8% in the solution stability test, slightly higher than the repeatability test (7%), but the difference was not significant, and stability was still maintained.

[0239] The results showed that the qPCR method used provided stable and consistent results across instruments and after short-term storage. Although the %CV of spike recovery increased in some cases, overall the robustness of the method and the stability of the sample solutions met the experimental requirements.

[0240] Example 10 Linearity Verification

[0241] This example verifies the linearity of the qPCR method and determines whether the method can accurately measure samples of different concentrations within a certain range. 2 The value and slope determine the linearity of the method.

[0242] The specific experimental plan is as follows:

[0243] Summarize the standard curve data obtained from previously performed repeatability, intermediate precision, accuracy, range, and limit of quantitation experiments.

[0244] Data Analysis:

[0245] Calculate the correlation coefficient (R) of the standard curve 2 ) and slope to assess the linearity of qPCR assays. The acceptance criteria were the standard curve R 2 ≥0.98, the slope is between -3.10 and -3.60. The specific results are shown in Table 18 below:

[0246] Table 18 Linear relationship data

[0247]

[0248] in conclusion:

[0249] In all experiment types, R² values ​​were close to or equal to 0.999, indicating that the response of the qPCR method was highly correlated with the target DNA concentration within the measurement range. Slope values ​​ranged from -3.351 to -3.357, with the exception of the range experiment, which had a slightly higher slope (-3.278), and the limit of quantitation experiment, which had a slightly lower slope (-3.335).

[0250] All experimental R² values ​​exceeded the acceptance criteria of 0.98, and the slopes were all within the acceptance range (-3.10 to -3.60), demonstrating that the qPCR method has excellent linearity. Although the slope of the range experiment was slightly higher, it did not affect the overall linearity judgment, and the slope of the quantitation limit experiment was slightly lower but still within the acceptance criteria. These results verify that the qPCR method can provide reliable and consistent quantitative results over a wide concentration range.

[0251] This comprehensive conclusion presents a series of examples to verify the effectiveness of E. coli DNA standards for host cell DNA residue detection (qPCR) in plasmid products. Example 1 confirms that the preparation quality of the standard meets the predetermined standards, and the concentration and purity meet the requirements. Subsequent Examples 2 to 10 comprehensively evaluate the accuracy, specificity, linearity and precision of the detection method through comparative analysis, specificity and sensitivity verification, and stability testing under different conditions. The method shows good repeatability and intermediate precision, as well as stability between different devices. In addition, the range and quantitative limit of the method have been clearly defined, further supporting its reliability in practical applications.

[0252] After the preparation of E.coli DNA standard, the results of concentration quantification and AGE detection were in line with the standards. E.coli DNA standard can be used as a standard for host cell DNA residue detection (qPCR) in plasmid products. The concentration of E.coli DNA standard is 27.5ng / μL, and the AGE result is a complete genome band without impurities. After comparing the results of the method with the commercial E.coli DNA residue detection reference kit, the %CV was less than 30%, and the results were good compared with the reference kit. After the method was verified, the experimental results all met the validity judgment criteria; the method has good specificity, linearity, accuracy, precision and durability between different detection instruments and solution placement; the range of the method is 300~0.03pg / μL, and the limit of quantification is 0.03pg / μL.

[0253] In summary, the host cell DNA residue detection (qPCR) method can be used for the detection of E. coli DNA residues in plasmid products. The qPCR detection method of the present invention can not only accurately measure the E. coli DNA residues in plasmid products, but also has all the key verification indicators required for laboratory and industrial applications.

[0254] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A specific primer and probe combination for qPCR reaction for detecting residual Escherichia coli DNA in a sample, wherein: The specific primer and probe combination comprises: The forward primer sequence is 5'-TGCTGGAAGGTAATCAAC-3', as shown in SEQ ID NO.1; The reverse primer sequence is 5'-GGGATTTCAAACGATCCTC-3', as shown in SEQ ID NO.2; The probe sequence is 5'-FAM-TCATTCTCACCTCGGATCGCT-BHQ1-3', as shown in SEQ ID NO.

3.

2. The specific primer and probe combination according to claim 1, wherein: The Escherichia coli DNA in the sample is the Escherichia coli dnaA gene, and the dnaA gene sequence is shown in SEQ ID NO.

4.

3. The specific primer and probe combination according to claim 1 or 2, wherein: The size of the amplified product of the specific primer and probe combination is 84 bp.

4. The specific primer and probe combination according to claim 1, wherein: The sample is selected from vaccines, proteins, nucleic acid fragments, plasmids or vitamins produced by Escherichia coli.

5. A qPCR reaction kit for detecting residual Escherichia coli DNA in a plasmid product, characterized in that: The kit comprises the specific primer and probe combination according to any one of claims 1 to 4.

6. The kit according to claim 5, wherein The kit also includes E. coli DNA standards and qPCR reaction solution.

7. The kit according to claim 6, wherein The qPCR reaction solution includes: dNTP mixed solution, nuclease RNase H, hot start PCR enzyme, Mg 2+ solution and nuclease-free water.

8. The kit according to claim 5, wherein The minimum detection limit of the kit for Escherichia coli DNA in plasmid products is 0.03 pg / μL.

9. The kit according to claim 5, wherein the detection range of the kit for Escherichia coli DNA in the plasmid product is 0.03 pg / μL to 300 pg / μL.

10. A qPCR method for detecting residual host cell DNA, characterized in that: The host cell is Escherichia coli, and the method is used to detect plasmid products, and the method comprises the steps of using the specific primer and probe combination described in any one of claims 1 to 4 or the kit described in any one of claims 5 to 9; The method comprises the following steps: a) preparing a series of standard solutions of E. coli DNA; b) preparing sample solution; c) Prepare spiked sample solution; d) preparing a qPCR mixed solution, comprising the step of mixing the components of the forward primer sequence, reverse primer sequence and probe sequence according to claim 1 with a qPCR reaction solution; e) Add samples and perform qPCR detection; f) Calculate and analyze the results.

11. Use of the specific primer and probe combination according to any one of claims 1 to 4, the kit according to any one of claims 5 to 9, or the method according to claim 10 for detecting and quantitatively analyzing residual host cell DNA in intermediates, semi-finished products, and finished products of vaccines, recombinant proteins, antibodies, cells, and gene drugs, wherein: The host cell is Escherichia coli.

Images