Detection of Extraneous Gene Residues in Products

Detection of Extraneous Gene Residues in Products

1. Background Introduction

With the rapid development of synthetic biology and genetic engineering technologies, genetically modified microorganisms (GMMs) have been widely applied in multiple fields such as biomedicine, food processing, industrial fermentation, and agricultural biotechnology. By introducing exogenous genes or modifying endogenous genes, GMMs can efficiently express target products, providing strong technical support for industrial development. Globally, the number of products such as biological products, enzymes, food additives, and novel food ingredients produced using GMMs continues to grow, and the market scale is expanding steadily.

However, the safety evaluation of GMM-derived products is a key prerequisite for the healthy development of the industry. Residual exogenous gene DNA in products may pose multiple potential risks, including horizontal gene transfer risk, oncogenic risk (containing oncogene sequences) [1], antibiotic resistance transmission risk (carrying resistance marker genes), and immunogenicity risk [2]. Therefore, strict control and detection of exogenous gene residues in products have become necessary measures to ensure product safety.

International regulatory requirements are increasingly stringent. The World Health Organization (WHO) clearly stipulates that residual DNA in biological products shall not exceed 10 ng per dose [3]. The United States Pharmacopeia (USP) provides detailed regulations on residual DNA detection methods and standards in General Chapters 0> and 9>, and designates quantitative PCR (qPCR) as the recommended standard method [4,5]. The European Pharmacopoeia (EP) also sets strict limit requirements for residual DNA.

The domestic regulatory framework is constantly improving. In September 2024, the National Center for Food Safety Risk Assessment issued the "Requirements for Application Materials for Safety Evaluation of Genetically Modified Microorganisms Used in Food Processing (Trial)", which explicitly requires applicants to provide "detection data of exogenous gene residues in products". PCR methods should be used to detect specific DNA fragments of production strains, with a detection threshold not higher than 10 ng DNA/g (mL) sample [6]. The "Guidelines for Identification and Safety Evaluation of Production Strains of Direct-Fed Microorganisms and Fermented Products" issued by the Ministry of Agriculture and Rural Affairs in 2021 also puts forward clear requirements for exogenous gene detection [7]. In addition, documents in the biopharmaceutical field such as the "Chinese Pharmacopoeia" and "Key Quality Control Points for Recombinant DNA Products for Human Use" have set strict regulations on residual DNA content [8].

Traditional residual DNA detection methods such as Southern blot, PicoGreen fluorescent dye method, and Threshold immunoassay have limitations including insufficient sensitivity, cumbersome operation, and low throughput, which can no longer meet the increasingly strict regulatory requirements. To address this industry pain point, Shutong Technology has developed a high-sensitivity exogenous gene residue detection platform based on quantitative PCR (qPCR) technology, providing reliable technical support for the safety evaluation of GMM-derived products.

2. Principles of Extraneous Gene Residue Detection

2.1 Detection Principle

The detection of exogenous gene residues in GMMs is based on the precise recognition and quantitative capability of qPCR technology for specific DNA sequences. Unlike traditional total DNA detection methods, qPCR technology targets specific exogenous gene sequences introduced during genetic modification, offering high specificity and sensitivity.

Figure 1. Schematic Diagram of qPCR Detection Principle

2.2 Detection Workflow

(1) Sample DNA Extraction: Total DNA is extracted from the test product. Optimized extraction methods are used to remove interfering substances such as proteins and polysaccharides, ensuring high recovery rate (70%-130%).

(2) Primer Design and Validation: Highly specific primer pairs are designed for specific sequences of exogenous genes, followed by specificity validation (including melting curve analysis and agarose gel electrophoresis) and sensitivity validation.

(3) Standard Curve Construction: A standard curve is established using exogenous gene standard DNA of known concentration (such as plasmid DNA or genomic DNA of production strains) to ensure the linear range and amplification efficiency meet requirements.

(4) qPCR Amplification and Real-Time Monitoring: Real-time amplification is performed on a quantitative real-time PCR instrument, and changes in SYBR Green fluorescence signals in each cycle are monitored in real time.

(5) Melting Curve Analysis: After amplification, melting curve analysis is performed to verify the specificity of amplification products and confirm the absence of primer dimers and non-specific amplification.

(6) Data Analysis and Quantification: The residual amount of exogenous genes in the sample is calculated based on the Ct value of the sample and the standard curve.

(7) Quality Control: Multiple quality control measures are set including positive control, negative control, no-template control (NTC), and PCR inhibition detection to ensure the accuracy and reliability of detection results.

3. Technological Innovations and Advantages of qPCR

3.1 Core Technological Innovations

3.1.1 Multi-Target Detection Strategy

A comprehensive detection scheme is designed for various exogenous gene elements that may be introduced during genetic modification:

(1) Target Gene Sequence Detection: Specific amplification of functional genes;

(2) Promoter and Terminator Detection: Detection of commonly used expression regulatory elements (such as T7, lac, CMV promoters);

(3) Marker Gene Detection: Detection of antibiotic resistance genes (such as kanamycin, ampicillin resistance genes) or reporter genes (such as GFP, RFP);

(4) Vector Backbone Sequence Detection: Detection of vector characteristic sequences such as plasmid origin of replication and multiple cloning sites.

3.1.2 High-Sensitivity Detection System

A high-sensitivity detection system is constructed by optimizing DNA extraction methods, primer design, and PCR reaction conditions:

(1) Optimized DNA extraction methods ensure high recovery rate (70%-130%), meeting pharmacopoeia requirements;

(2) Specific primer design avoids non-specific amplification and primer dimer formation;

(3) Optimized SYBR Green reaction system improves amplification efficiency (90%-110%) and detection sensitivity;

(4) Melting curve analysis ensures the specificity of amplification products, effectively eliminating false positives.

3.1.3 Strict Quality Control System

Multiple quality control measures are established to ensure the accuracy and reliability of detection results:

(1) Positive Control: Genomic DNA of production strains is used as positive control;

(2) Negative Control: DNA without target genes or no-template control (NTC) is used to exclude contamination;

(3) Melting Curve Validation: Melting curve analysis is performed for each sample to confirm the specificity of amplification products;

(4) PCR Inhibition Exclusion: Standard DNA of known concentration is added to sample DNA to verify PCR reaction efficiency;

(5) At least 3 technical replicates are performed for each sample to ensure data precision (standard deviation 5).

3.2 Method Validation and Performance Indicators

In accordance with ICH Q2 (R1), Chinese Pharmacopoeia, WHO, and FDA relevant guidelines, Shutong Technology has completed comprehensive and systematic method validation:

4. Application Scenarios and Service Advantages

4.1 Wide Application Scenarios

Biomedical Field:

(1) Detection of host cell DNA and exogenous gene residues in biological products such as recombinant protein drugs, monoclonal antibodies, and vaccines;

(2) Detection of vector DNA residues in cell and gene therapy products;

(3) Exogenous DNA residue data support for IND/NDA filings.

Food Industry Field:

(1) Detection of exogenous gene residues in novel food ingredients and food additives produced using GMMs;

(2) Exogenous gene detection in food enzymes (such as α-amylase, protease);

(3) Support for applications of "three new foods" (novel food ingredients, new varieties of food additives, new varieties of food-related products).

Industrial Fermentation Field:

(1) Detection of exogenous gene residues in fermentation products such as industrial enzymes, organic acids, and amino acids;

(2) Safety evaluation of production strains for bio-based chemicals.

Agricultural Biotechnology Field:

(1) Detection of exogenous gene residues in microbial fertilizers, biopesticides, and feed additives;

(2) Safety evaluation of direct-fed microbial products.

4.2 Service Advantages

(1) Advanced Technology: Adopts internationally mainstream SYBR Green qPCR detection technology, complying with international and domestic regulatory requirements such as WHO, FDA, and Chinese Pharmacopoeia. It features high detection sensitivity, strong specificity, accurate quantification, and significant cost-effectiveness.

(2) Complete Qualifications: The laboratory is certified by ISO 9001 and CNAS quality management systems, ensuring data reliability and complete traceability.

(3) Comprehensive Method Validation: Has completed comprehensive method validation in line with ICH Q2(R1) standards (specificity, sensitivity, linear range, accuracy, precision, robustness). The validation report can directly support IND/NDA filings and "three new foods" applications.

(4) Customized Services: Customizes primer design and establishes targeted detection methods based on strain information and exogenous gene sequences provided by clients.

(5) Fast and Efficient: Standard detection cycle is 7-10 working days; urgent projects can be processed expeditedly.

(6) One-Stop Solution: Provides full-process services from sample DNA extraction, qPCR detection, data analysis to report issuance. Meanwhile, supporting services such as viable bacteria residue detection, strain identification, and whole-genome sequencing are available.

(7) Professional Technical Support: An experienced technical team provides comprehensive technical services including experimental design consultation, data interpretation, and filing support.

5. Core Content of Extraneous Gene Residue Detection Example Report

Shutong Technology provides comprehensive exogenous gene residue detection reports compliant with regulatory requirements, including the following core content:

(1) Sample Information and Detection Methods: Detailed records of sample number, batch, detection date, adopted detection method (SYBR Green qPCR), primer sequences, amplicon length, and other information.

(2) Standard Curve and Amplification Curve:

Standard Curve: Displays the linear relationship between Ct values of standard DNA at different concentrations and log DNA concentration, indicating slope, intercept, correlation coefficient R², and amplification efficiency;

Amplification Curve: Displays real-time fluorescence amplification curves of standards and samples to verify the effectiveness of PCR reactions.

(3) Melting Curve Analysis: Displays the melting curve of PCR products to verify their specificity. A single sharp peak indicates good specificity of amplification products, with no primer dimers or non-specific amplification. The melting temperature (Tm value) should be within the expected range, and Tm values of different samples should be consistent (±0.5°C).

(4) Summary Table of Detection Results: Presents detection results of each sample in tabular form:

(5) Method Validation Data (Optional, for Filing Support):

1. Specificity Validation: Primer sequence information, amplification product sequencing results, melting curve analysis;

2. Sensitivity Validation: Limit of Detection (LOD) and Limit of Quantification (LOQ) determination data;

3. Linear Range Validation: Standard curve data;

4. Accuracy Validation: Spiked recovery rate data;

5. Precision Validation: Intra-batch and inter-batch repeatability data.

(6) Conclusion and Recommendations: Provides a clear conclusion based on detection results, indicating whether the sample meets relevant regulatory requirements (e.g., ≤10 ng DNA/g), and offers technical recommendations as needed.

6. Extraneous Gene Residue Detection Service Content

Standard Service Cycle: 7-10 working days (calculated from the date of qualified sample quality inspection); urgent projects can be processed expeditedly through negotiation.

7. Sample Requirements

8. References

[1] Sheng, L., Cai, F., Zhu, Y., Pal, A., Athanasiou, M., Orrison, B., Blair, D. G., Hughes, S. H., Coffin, J. M., Lewis, A. M., & Peden, K. (2008). Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H-ras and c-myc. Biologicals, 36(3), 184-197.

[2] Wang, X., Morgan, D. M., Wang, G., & Mozier, N. M. (2012). Residual DNA Analysis in Biologics Development: Review of Measurement and Quantitation Technologies and Future Directions. Biotechnology and Bioengineering, 109(2), 307-317.

[3] World Health Organization. (1998). Requirements for the use of animal cells as in vitro substrates for the production of biologicals. WHO Technical Report Series, No. 878, Annex 1.

[4] United States Pharmacopeia. General Chapter 30> Nucleic Acid-Based Techniques—Approaches for Detecting Trace Nucleic Acids (Residual DNA Testing). USP-NF.

[5] United States Pharmacopeia. General Chapter <509> Residual DNA Testing. USP-NF.

[6]国家食品安全风险评估中心.(2024).食品加工用遗传修饰微生物安全性评价申报材料要求(试行),附件1.

[7]农业农村部.(2021).直接饲喂微生物和发酵制品生产菌株鉴定及其安全性评价指南.农业农村部公告第485号.

[8]国家药典委员会.(2020).中华人民共和国药典(2020年版)四部,通则1143外源性DNA残留量测定法.