Real-Time PCR in Microbiology: Principles and Applications

Real-time polymerase chain reaction (real-time PCR) is a molecular technique that monitors DNA amplification during the reaction, enabling quantification of target nucleic acids without post-PCR processing. This article explains the core principles of real-time PCR chemistries, including SYBR Green and TaqMan probe systems, and compares their applications in microbial detection and quantification for laboratory students, technicians, researchers, and diagnostic professionals. Real-time quantitative PCR is broadly classified into two types based on its purpose: absolute and relative quantification [3]. Absolute quantification is used in microbiology, food technology, and biotechnology to quantify microbial load or copy numbers, whereas relative quantification is used in genomics and functional transcriptomics for gene expression analysis [3]. The technique allows direct detection of PCR product during the exponential phase of the reaction, providing a powerful, accurate, rapid, and sensitive method for detection of microorganisms, differentiation of species, quantification of microbial load, and detection of drug resistance [8].

At a Glance: Real-Time PCR Chemistries and Applications

Core Principles of Real-Time PCR

Fluorescence Monitoring During Amplification

Real-time PCR instruments measure fluorescence at each cycle of the PCR reaction. The fluorescence signal increases proportionally with the accumulation of PCR product during the exponential phase of amplification [8]. The cycle at which fluorescence crosses a defined threshold (Cq or Ct value) is inversely proportional to the initial target quantity. This relationship allows accurate quantification over a wide dynamic range, typically 5 to 7 log10 units.

Absolute and Relative Quantification

Absolute quantification determines the exact copy number of a target sequence in a sample by comparing Cq values to a standard curve generated from known concentrations of a reference standard [3]. This approach is used to quantify microbiological load in food samples, environmental samples, and clinical specimens. Relative quantification measures changes in target abundance relative to a reference sample, commonly used for gene expression analysis where fold-change is reported [3].

PCR Efficiency and Standard Curves

PCR efficiency is a critical parameter that affects quantification accuracy. A standard curve is generated by amplifying serial dilutions of a known template and plotting Cq values against log template concentration. The slope of the standard curve indicates PCR efficiency, with a slope of -3.32 corresponding to 100% efficiency. Efficiency values between 90% and 110% are generally acceptable for reliable quantification. Primer characteristics, including melting temperature, GC content, and secondary structure, directly influence PCR efficiency and must be optimized for each assay [3].

SYBR Green Chemistry

Mechanism of Detection

SYBR Green is a fluorescent dye that binds to the minor groove of double-stranded DNA. When unbound, the dye exhibits minimal fluorescence. Upon binding to double-stranded DNA, fluorescence increases dramatically. During PCR, fluorescence accumulates as double-stranded amplicon is produced. At the end of each cycle, fluorescence is measured, and the increase correlates with the amount of PCR product.

Melt Curve Analysis for Specificity Verification

A key advantage of SYBR Green chemistry is the ability to perform melt curve analysis after amplification. As the temperature is gradually increased, double-stranded DNA denatures, releasing the dye and causing a drop in fluorescence. The melting temperature (Tm) is characteristic of each amplicon based on its length and GC content. Specific amplicons show a distinct Tm that differs from non-specific products or primer-dimers [4]. For example, in a genotyping assay for SERPINA1 gene variants, specific amplicons showed a difference of 2.0 degrees Celsius in melting temperature for non-S and S variants and of 2.9 degrees Celsius for non-Z and Z variants [4]. This allows clear discrimination of mutant and wild-type variants.

Applications in Genotyping and Pathogen Detection

SYBR Green-based real-time PCR is used for genotyping applications where allele-specific primers are combined with melt curve analysis. The method is robust, fast, and easily scalable on standard real-time PCR platforms [4]. It overcomes the handicaps of non-homogeneous approaches and greatly reduces genotyping costs compared with other homogeneous approaches [4]. In microbiology, SYBR Green assays are used for detection and quantification of bacteria, fungi, and viruses where high specificity is not required or where melt curve analysis can distinguish targets.

Limitations and Quality Control Considerations

SYBR Green binds to any double-stranded DNA, including primer-dimers and non-specific amplification products. This can lead to false-positive signals if melt curve analysis is not performed. Quality control measures include including no-template controls, verifying melt curves for each sample, and confirming amplicon size by gel electrophoresis when establishing new assays. The inability to multiplex with SYBR Green limits its use in assays requiring simultaneous detection of multiple targets.

TaqMan Probe Chemistry

Mechanism of Detection

TaqMan probes are oligonucleotides labeled with a reporter fluorophore at the 5' end and a quencher at the 3' end. When the probe is intact, the quencher absorbs fluorescence from the reporter through fluorescence resonance energy transfer. During PCR extension, the 5' to 3' exonuclease activity of Taq polymerase cleaves the probe, separating the reporter from the quencher and generating fluorescence. Fluorescence increases with each cycle as more probe is cleaved, and the signal is proportional to the amount of target amplicon.

Multiplex Capabilities

TaqMan probes can be labeled with different fluorophores with distinct emission spectra, allowing simultaneous detection of multiple targets in a single reaction. Multiplex real-time PCR assays can detect and differentiate multiple pathogens, such as Campylobacter jejuni and Campylobacter coli, by targeting species-specific genes with different probes [5]. In a multiplex assay for Campylobacter detection, species differentiation was done by targeting mapA for C. jejuni, ceuE for C. coli, and cje for both species [5]. The detection limit of the multiplex real-time PCR assay was 4.5 and 5.5 log10 cfu/mL in enrichment broth and broth containing chicken skin, respectively [5].

Applications in Clinical Diagnostics

TaqMan-based real-time PCR is widely used in clinical microbiology for detection and quantification of pathogens. The technique provides high sensitivity and specificity, with limits of detection ranging from 1 to 100 copies per reaction depending on the target [6]. For SARS-CoV-2 variant detection, a multiplex real-time PCR based on target failure principle accurately detected multiple variants that existed in a single sample, with limits of detection ranging from 1 to 10 copies for Omicron BA.2 and BA.5, and from 10 to 100 copies for Delta and other variants [6]. The method enables rapid and timely detection of existing and future variants for epidemiological surveillance and diagnostic applications [6].

Commercial Assay Performance

Commercial real-time PCR tests based on multiplex probe amplification technology provide simultaneous detection and differentiation of multiple genotypes in a single closed-tube reaction, ensuring rapid, cost-effective, and contamination-free results [18]. For example, the Papilloplex HR-HPV assay detects and differentiates 14 high-risk human papillomavirus genotypes in a single reaction with robust analytical performance and reproducible results [18]. These assays undergo rigorous validation including sensitivity, specificity, range, reproducibility, and cross-reactivity testing [18].

Practical Workflow for Real-Time PCR

Sample Preparation and Nucleic Acid Extraction

Nucleic acid extraction quality directly affects real-time PCR results. Template nucleic acid quality and extraction efficiency are among the limitations that can affect the validity of quantification data [7]. For microbial detection from food or environmental samples, enrichment steps may be required to increase target concentration to detectable levels. For Campylobacter detection from food, enrichment in Bolton broth combined with multiplex real-time PCR takes about 2 days to produce reliable results, while conventional culture methods can take up to 8 days [5]. A Monte Carlo simulation predicted that cold-stressed campylobacters could reach the detection limit after 40 hours of enrichment [5].

Primer and Probe Design

Primer and probe design is critical for assay specificity and efficiency. Key considerations include melting temperature (typically 58-60 degrees Celsius for primers, 68-70 degrees Celsius for probes), GC content (40-60%), amplicon length (70-150 base pairs for optimal efficiency), and avoidance of secondary structure and primer-dimer formation. For multiplex assays, primers and probes must be designed to have compatible melting temperatures and minimal cross-reactivity. For target failure-based assays, primers and probes are designed based on specific deletion mutations of circulating variants [6].

Reaction Setup and Thermal Cycling Conditions

Standard real-time PCR reactions include template DNA, primers, probes or SYBR Green dye, DNA polymerase, nucleotides, and buffer. Thermal cycling conditions typically include an initial denaturation step (95 degrees Celsius for 2-10 minutes), followed by 35-45 cycles of denaturation (95 degrees Celsius for 10-30 seconds), annealing (55-65 degrees Celsius for 20-60 seconds), and extension (72 degrees Celsius for 20-60 seconds). For TaqMan assays, annealing and extension are often combined at 60 degrees Celsius. For SYBR Green assays, a melt curve step is added after amplification, typically ramping from 65 to 95 degrees Celsius with fluorescence measurements every 0.5-1 degree Celsius.

Data Analysis and Interpretation

Data analysis involves setting the threshold line, determining Cq values for each sample, and calculating target quantities. For absolute quantification, a standard curve is generated from serial dilutions of a known standard. The Cq values of unknown samples are interpolated from the standard curve to determine copy numbers. For relative quantification, the delta-delta Cq method is used to calculate fold-change relative to a reference sample and a reference gene. PCR efficiency must be calculated from the standard curve slope, and efficiency correction should be applied if efficiency deviates significantly from 100% [3].

Options and Tradeoffs in Real-Time PCR Chemistries

SYBR Green vs. TaqMan: Selection Criteria

The choice between SYBR Green and TaqMan depends on the specific application, budget, and required specificity. SYBR Green is suitable for applications where cost is a primary concern, where melt curve analysis can provide specificity, or where multiple targets are not required. TaqMan is preferred for multiplex assays, clinical diagnostics requiring high specificity, and applications where quantification accuracy is critical. For genotyping applications, SYBR Green with melt curve analysis provides a cost-effective alternative to probe-based methods, with clear discrimination of variants based on melting temperature differences [4].

Multiplex Assay Design Considerations

Multiplex real-time PCR assays require careful optimization to ensure balanced amplification of all targets. Factors to consider include fluorophore selection to minimize spectral overlap, primer and probe concentration optimization to avoid competition, and verification of no cross-reactivity between targets. For pathogen detection, multiplex assays can simultaneously detect multiple species or variants, reducing time and cost compared to single-plex assays [5, 6]. The detection limit may vary between targets in a multiplex assay, and validation should include assessment of each target individually and in combination.

Viability Discrimination with Propidium Monoazide

A primary limitation of real-time PCR is its inability to differentiate between living and dead bacterial cells [11]. Propidium monoazide (PMA) is a viability dye that selectively penetrates damaged cell membranes and binds to DNA, preventing amplification of DNA from non-viable cells. PMA combined with real-time PCR (PMA-qPCR) enables exclusive detection of viable cells in food samples [11]. Multiplex PMA-qPCR demonstrated statistically significant agreement with gold-standard culture methods for quantifying Escherichia coli and Staphylococcus aureus in food matrices including cheese, frozen beef burgers, and raw oyster fillets [11]. For Salmonella detection, multiplex PMA-qPCR was more sensitive than microbiological culture methods [11]. PMA-qPCR represents a promising alternative method for enhanced pathogen detection in foods, offering reduced analysis time and decreased non-viable cell amplification [11].

Observations and Measurements in Real-Time PCR

Standard Curve Parameters

Standard curves are generated by amplifying serial dilutions of a known template, typically 10-fold dilutions covering 5-7 log10 units. Key parameters include slope (indicating efficiency), y-intercept (indicating sensitivity), R-squared value (indicating linearity), and dynamic range. Acceptable standard curves have slopes between -3.1 and -3.6 (90-110% efficiency), R-squared values greater than 0.98, and consistent amplification across the dynamic range. For multiplex assays, standard curves should be generated for each target individually and in combination to assess potential interference.

Limit of Detection and Limit of Quantification

The limit of detection is the lowest concentration of target that can be reliably detected, typically defined as the concentration at which 95% of replicates are positive. The limit of quantification is the lowest concentration that can be quantified with acceptable precision and accuracy. For real-time PCR assays, limits of detection can range from 1 to 100 copies per reaction depending on the target and assay design [6, 13]. For example, a multiplex qPCR assay for bovine viral diarrhea virus and pathogenic Escherichia coli achieved limits of detection of 10^2 copies per microliter for BVDV and 10^1 copies per microliter for E. coli K99 plasmid DNA [13].

Reproducibility and Repeatability

Reproducibility is assessed by running the same samples on different days, by different operators, or on different instruments. Repeatability is assessed by running replicates within the same run. Acceptable coefficients of variation for Cq values are typically less than 5% for replicate samples and less than 10% for inter-run comparisons. For droplet digital PCR, relative standard deviation for reproducibility validation was 0.77% [15]. Regular quality control samples should be included in each run to monitor assay performance over time.

Records and Documentation

Essential Records for Real-Time PCR Assays

Laboratories performing real-time PCR should maintain records of assay validation, including standard curve parameters, limit of detection, limit of quantification, specificity testing, and reproducibility data. For each run, records should include sample identification, extraction method and date, reaction setup details (reagent lots, concentrations), thermal cycling conditions, instrument used, Cq values for all samples and controls, standard curve data, and any deviations from standard protocols. For diagnostic applications, records should also include interpretation of results and any follow-up actions.

Quality Control Documentation

Quality control records should include results from no-template controls, positive controls, negative controls, and internal amplification controls. For each control, acceptable ranges for Cq values should be defined, and any out-of-range results should be documented with corrective actions taken. Regular monitoring of control results allows early detection of reagent degradation, instrument malfunction, or contamination issues. For quantitative assays, standard curve parameters should be tracked over time to monitor assay stability.

Common Failure Patterns and Troubleshooting

No Amplification or Late Amplification

Failure to amplify or late amplification can result from degraded template DNA, inhibitors in the sample, incorrect reaction setup, or instrument malfunction. Troubleshooting steps include verifying template quality by spectrophotometry or gel electrophoresis, running a positive control to confirm reagent and instrument function, checking primer and probe sequences for errors, and testing for inhibitors by spiking known template into the sample. For samples with suspected inhibitors, dilution of the template or additional purification steps may resolve the issue.

Non-Specific Amplification

Non-specific amplification appears as multiple peaks in melt curve analysis for SYBR Green assays or unexpected amplification in negative controls. Causes include primer-dimer formation, mispriming due to low annealing temperature, or contamination. Solutions include redesigning primers to avoid secondary structure, increasing annealing temperature, optimizing primer concentrations, and including no-template controls in each run. For SYBR Green assays, melt curve analysis should be performed to distinguish specific from non-specific products [4].

Poor Efficiency or Nonlinear Standard Curves

Poor PCR efficiency (slope outside -3.1 to -3.6) or nonlinear standard curves can result from suboptimal primer design, incorrect template dilution, or pipetting errors. Troubleshooting includes verifying primer sequences and melting temperatures, preparing fresh serial dilutions of the standard, ensuring accurate pipetting, and optimizing annealing temperature. For multiplex assays, primer and probe concentrations may need adjustment to balance amplification of all targets.

Fluorescence Signal Variability

High variability in fluorescence signals between replicates can result from pipetting errors, uneven temperature distribution in the thermal cycler, or photobleaching of fluorophores. Solutions include using master mixes to reduce pipetting steps, ensuring proper plate sealing, verifying instrument calibration, and using appropriate fluorophore concentrations. For multiplex assays, spectral overlap between fluorophores can cause signal variability and should be corrected by color compensation.

Limitations of Real-Time PCR

Template Quality and Extraction Efficiency

Template nucleic acid quality and extraction efficiency are critical limitations that can affect the validity of quantification data [7]. Incomplete lysis of microbial cells, degradation of nucleic acids during extraction, or co-extraction of inhibitors can lead to underestimation of target quantity. Standardization of extraction protocols and inclusion of internal amplification controls are essential for reliable results. For environmental samples, humic acids and other organic compounds can inhibit PCR and require additional purification steps.

Amplification of Nonviable DNA

Real-time PCR cannot differentiate between DNA from viable and nonviable cells, which can lead to overestimation of microbial load in samples where dead cells are present [7, 11]. This is particularly relevant for food safety applications where only viable pathogens pose a risk. PMA treatment can reduce but not eliminate amplification from nonviable cells, and the effectiveness of PMA depends on the type of sample and the condition of the cells [11].

Gene Copy Number Variation

Quantification based on single-copy genes may not accurately reflect cell numbers if gene copy number varies between strains or species [7]. For example, bacteria can have multiple copies of rRNA genes, and the copy number can vary between species and growth conditions. Using multicopy target genes can increase sensitivity but may complicate quantification. For accurate quantification, the target gene copy number per genome should be known and accounted for in calculations.

Limited Number of Sequences in Databases

The specificity of real-time PCR assays depends on the availability of sequence data for primer and probe design. For emerging pathogens or poorly characterized organisms, limited sequence information can lead to assays that fail to detect all variants or cross-react with related species [7]. Regular monitoring of sequence databases and updating of assay designs are necessary to maintain specificity.

Safety and Regulatory Context

Biosafety Considerations for Real-Time PCR

Real-time PCR should be performed in a laboratory setting that follows appropriate biosafety practices as outlined in the Laboratory Biosafety Manual from the World Health Organization [1]. Sample handling, nucleic acid extraction, and reaction setup should be conducted in designated areas with appropriate containment for the microorganisms being tested. For clinical samples or samples containing known pathogens, work should be performed in a biosafety cabinet. Amplification products should be handled as potential contaminants, and post-PCR analysis areas should be physically separated from pre-PCR areas to prevent carryover contamination.

Regulatory Framework for Diagnostic Assays

Real-time PCR assays used for clinical diagnostics must comply with applicable regulatory requirements, including validation of analytical performance characteristics such as sensitivity, specificity, accuracy, precision, and reproducibility. Commercial assays may be CE-IVD marked or FDA cleared for specific applications [18]. Laboratory-developed tests require in-house validation and should follow guidelines from regulatory authorities. For food safety applications, methods may be validated against reference standards such as ISO methods [5].

Quality Assurance and Proficiency Testing

Laboratories performing real-time PCR should participate in proficiency testing programs to verify the accuracy of their results. Internal quality control measures include use of positive and negative controls, replicate testing, and monitoring of control results over time. For quantitative assays, standard curves should be verified regularly, and instrument calibration should be performed according to manufacturer recommendations. Documentation of all quality control activities is essential for accreditation and regulatory compliance.

Professional Escalation Criteria

When to Seek Technical Support

Technical support should be sought when troubleshooting fails to resolve persistent issues such as no amplification, high variability, or poor efficiency. Instrument malfunction, such as uneven temperature distribution or optical system failure, requires manufacturer service. For assay design issues, consultation with experienced molecular biologists or the assay manufacturer may be necessary. If contamination is suspected and cannot be eliminated, laboratory procedures should be reviewed and potentially redesigned.

When to Refer Samples to Reference Laboratories

Samples should be referred to reference laboratories when results are inconclusive or contradictory, when the assay is not validated for the specific sample type, or when confirmation by an alternative method is required. For emerging pathogens or unusual results that may indicate a novel variant, reference laboratories with sequencing capabilities can provide definitive identification. For legal or regulatory purposes, results may need to be confirmed by a reference laboratory using validated methods.

When to Report Results to Public Health Authorities

Positive results for notifiable pathogens should be reported to public health authorities according to local regulations. Laboratories should have protocols in place for timely reporting of results that may have public health implications. For food safety applications, positive results for pathogens such as Salmonella, Campylobacter, or pathogenic E. coli should be reported to food safety authorities to enable traceback and recall actions.

Frequently Asked Questions

What is the difference between real-time PCR and conventional PCR?

Real-time PCR monitors amplification during the reaction by measuring fluorescence at each cycle, allowing quantification without post-PCR processing. Conventional PCR requires gel electrophoresis or other detection methods after amplification and provides only qualitative or semi-quantitative results. Real-time PCR is faster, more sensitive, and provides accurate quantification over a wide dynamic range [8].

How do I choose between SYBR Green and TaqMan for my application?

Choose SYBR Green when cost is a primary concern, when melt curve analysis can provide specificity, or when developing new assays for novel targets. Choose TaqMan when high specificity is required, when multiplexing multiple targets, or for clinical diagnostic applications where accuracy is critical. SYBR Green is suitable for gene expression analysis and genotyping with melt curve analysis, while TaqMan is preferred for pathogen detection and quantification in complex samples [3, 4].

What is the purpose of melt curve analysis in SYBR Green real-time PCR?

Melt curve analysis verifies the specificity of amplification by measuring the melting temperature of the PCR product. Specific amplicons have characteristic melting temperatures based on their length and GC content. Non-specific products, such as primer-dimers, have different melting temperatures and can be distinguished from the target amplicon. Melt curve analysis is essential for SYBR Green assays to confirm that fluorescence signals are from the intended target [4].

How do I determine PCR efficiency from a standard curve?

PCR efficiency is calculated from the slope of the standard curve using the formula: Efficiency = 10^(-1/slope) - 1. A slope of -3.32 corresponds to 100% efficiency. Acceptable efficiency values range from 90% to 110% (slope between -3.1 and -3.6). Low efficiency may indicate suboptimal primer design, inhibitors, or incorrect reaction conditions. Efficiency should be calculated for each target in multiplex assays [3].

Can real-time PCR distinguish between live and dead microorganisms?

Standard real-time PCR cannot distinguish between live and dead microorganisms because DNA from dead cells can still be amplified. Propidium monoazide (PMA) treatment can selectively prevent amplification of DNA from dead cells by penetrating damaged cell membranes and binding to DNA. PMA-qPCR has been shown to provide results comparable to culture methods for quantifying viable bacteria in food samples [11].

What are the main limitations of real-time PCR for environmental samples?

Main limitations include template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database [7]. Environmental samples often contain inhibitors such as humic acids that can reduce PCR efficiency. Internal amplification controls should be used to detect inhibition, and extraction protocols should be optimized for each sample type.

How do I set up a multiplex real-time PCR assay?

Multiplex assay setup requires careful selection of fluorophores with minimal spectral overlap, design of primers and probes with compatible melting temperatures, and optimization of primer and probe concentrations to ensure balanced amplification of all targets. Validation should include assessment of detection limits for each target individually and in combination, verification of no cross-reactivity between targets, and confirmation of specificity against related organisms [5, 6].

What quality controls should be included in each real-time PCR run?

Each run should include a no-template control to detect contamination, a positive control to confirm reagent and instrument function, and a negative control to verify specificity. For quantitative assays, a standard curve should be included for absolute quantification. Internal amplification controls should be added to each sample to detect inhibition. Replicate testing of samples allows assessment of precision. Control results should be within predefined acceptable ranges for the run to be valid.

Related Guides

• Digital Droplet PCR (ddPCR) for Absolute Quantification of Viral Load in Veterinary Diagnostics: Principles and Applications
• Digital Droplet PCR (ddPCR) for Absolute Quantification of Viral Load in Veterinary Pathogens: Principles and Applications
• Real-Time PCR Assay for Detection of Bovine Respiratory Syncytial Virus and Pasteurella multocida in Cattle
• Nested PCR: Principles, Protocol, and Applications
• Quantitative Real-Time PCR for Detection of Feline Coronavirus Mutants Associated with Feline Infectious Peritonitis

References and Further Reading

• Laboratory Biosafety Manual. World Health Organization.
• NCBI Literature Resources. National Center for Biotechnology Information.
• Real-time quantitative PCR: A tool for absolute and relative quantification.. Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 2021.
• Real-time PCR detection of PIS and PIZ alleles of SERPINA1 gene using SYBR green.. Gene, 2024.
• Combining enrichment with multiplex real-time PCR leads to faster detection and identification of Campylobacter spp. in food compared to ISO 10272-1:2017.. Food microbiology, 2022.
• Multiplex RT Real-Time PCR Based on Target Failure to Detect and Identify Different Variants of SARS-CoV-2: A Feasible Method That Can Be Applied in Clinical Laboratories.. Diagnostics (Basel, Switzerland), 2023.
• Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: applications and considerations.. Biotechnology advances, 2013.
• Applications of real-time PCR technology to mycobacterial research.. The Indian journal of medical research, 2006.
• Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes.. Journal of fungi (Basel, Switzerland), 2022.
• Quantitative molecular methods in virology.. Archives of virology, 1995.
• Real-time quantitative PCR combined with propidium monoazide for simultaneous detection and quantification of Escherichia coli, Staphylococcus aureus and Salmonella spp. during processing of animal-based food products.. 2026.
• Progress of Rapid Detection Technology for Aquatic Microorganisms: A Comprehensive Review.. 2026.
• Development of a multiplex fluorescent qPCR assay for the simultaneous detection of bovine viral diarrhea virus and pathogenic Escherichia coli.. 2026.
• Editorial for Special Issue "Microbial Safety and Beneficial Microorganisms in Foods".. 2026.
• A droplet digital PCR method for the detection of scale drop disease virus in yellowfin seabream (Acanthopagrus latus). Frontiers in Microbiology, 2024.
• Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Frontiers in Microbiology, 2015.
• Research progress on nucleic acid amplification-based detection technologies for phytopathogenic fungi. Applied Microbiology and Biotechnology, 2026.
• Principles and analytical performance of Papilloplex® HR-HPV, a new commercial CE-IVD molecular diagnostic test for the detection of high-risk HPV genotypes.. Diagnostic microbiology and infectious disease, 2019.
• Targeting effect of berberine on type I fimbriae of Salmonella Typhimurium and its effective inhibition of biofilm. Applied Microbiology and Biotechnology, 2021.
• Advanced sample preparation for the molecular quantification of Staphylococcus aureus in artificially and naturally contaminated milk.. Journal of food microbiology, 2011.
• An overview of real-time quantitative PCR: Principles and formats for environmental microbiology studies. Asian Journal of Microbiology Biotechnology and Environmental Sciences, 2018.
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• Real-Time Detection of Amplification Products Through Fluorescence Quenching or Energy Transfer. Advanced Techniques in Diagnostic Microbiology Volume 1 Techniques Third Edition, 2018.

This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.