Rapid Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus by Multiplex Real-Time Fluorescent PCR

Summary

This protocol uses multiplex real-time PCR with primers/probes targeting SA-specific nuc and MRSA mecA genes. It enables rapid (<1 h), qualitative detection of SA/MRSA nucleic acids in human sputum samples, overcoming limitations of culture-based methods.

Abstract

Staphylococcus aureus (S.aureus) is a common Gram-positive human pathogen that causes community-acquired and nosocomial infections, with diverse clinical manifestations ranging from local superficial lesions and food poisoning to fatal systemic infections. The discovery of antibiotics significantly reduced the mortality rate, but the problem of drug resistance has since become increasingly prominent. Since the first identification of MRSA in 1960, this strain has emerged as a global public health threat. MRSA is a major pathogen of nosocomial infections, capable of causing various severe diseases such as endocarditis, chronic osteomyelitis, pneumonia, pyogenic arthritis, and bacteremia. Therefore, rapid and accurate detection of S. aureus and its drug resistance is crucial for guiding clinical treatment.

Current routine detection methods for S.aureus and MRSA have significant limitations. The traditional bacterial culture method, serving as the "gold standard" for decades, can provide definitive species identification and drug sensitivity results, but the process is time-consuming, taking 48 to 72 h. Furthermore, this method is susceptible to contamination and relies on specialized laboratory facilities and skilled technicians. Serological testing achieves non-invasive diagnosis by detecting S.aureus antibodies in patient serum, but it cannot distinguish between active infections and past infections, nor can it identify drug-resistant strains (such as MRSA).

This study focuses on developing a novel multiplex real-time fluorescence PCR detection method to overcome the above-mentioned limitations. This method designs specific primers and TaqMan fluorescent probes targeting the species-specific nuc gene of S.aureus and the mecA gene mediating methicillin resistance, enabling simultaneous amplification and detection of S.aureus and MRSA in a single reaction system. This technology greatly reduces the detection time, providing a rapid, accurate, and cost-effective solution for S.aureus and MRSA detection. This innovative approach greatly improves clinical diagnostic efficiency and facilitates the early implementation of targeted antibiotic therapy, making important contributions to controlling drug-resistant bacterial infections.

Introduction

S. aureus is a widespread Gram-positive conditional pathogen that colonizes the nasal mucosa of healthy individuals^1,2. When host immunity is compromised, it can cause local skin infections, deep tissue invasion, or systemic diseases (e.g., pneumonia, endocarditis, osteomyelitis, and bacteremia)³. Methicillin, a semisynthetic penicillin, was clinically applied for S. aureus infections in 1959, and two years later, MRSA emerged in the UK, rapidly spreading to European countries including Denmark, France, and Switzerland⁴. MRSA resistance to β-lactam antibiotics not only complicates clinical management but also significantly elevates infection risks⁵. Multiple factors-including uncontrollable colonization/spread, high prevention costs, and antibiotic overuse-have collectively driven up MRSA infection rates⁶. Owing to its rapid transmission and complex resistance mechanisms, MRSA infections are associated with high case-fatality rates, with nearly 150,000 reported cases and >7,000 deaths annually in EU countries⁷. According to CHINET surveillance data, MRSA infection rates in China have remained above 30% for the past five years⁸. The high risk of nosocomial cross-infection further intensifies public health burdens and strains healthcare systems^9,10. Therefore, the development of efficient and precise rapid MRSA detection technologies is clinically urgent for achieving early diagnosis, personalized therapy, reducing mortality, and interrupting transmission chains.

Current detection technologies for S.aureus and MRSA span multiple fields including phenotypic detection, genomic detection, transcriptomic detection, proteomic detection, mass spectrometry technology, and bioinformatics-based analyses¹¹.

Phenotypic detection methods, such as the disk diffusion method (KB method) and minimum inhibitory concentration method (MIC method), are recommended as "gold standards" by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These methods have the advantages of no need for specialized instruments, being widely used for resistance spectrum surveillance and epidemiological investigation. However, their disadvantages are also evident: long culture periods (over 24 h), and the results of the KB method for some new antibiotics require validation by MIC values, with results for breakpoint strains being susceptible to human factors^12,13.

Among genomic-level detection methods, conventional PCR has the characteristics of high sensitivity and strong specificity, enabling preliminary judgment of resistance and shortening experimental time, with low cost and high efficiency. But its limitations lie in being able to detect only known resistance genes and relying on specialized instruments¹⁴. The mechanism of microbial resistance is highly complex, making it crucial to develop technical methods that can cover unknown resistant phenotypes and enable rapid identification. The emerging GoPhAST-R (Combined genotypic and phenotypic AST through RNA detection) technology determines resistance phenotypes by detecting mRNA expression levels, enabling simultaneous detection of different resistance-related genes across multiple pathogens, shortening experimental turnaround time, and achieving 94-99% accuracy when combined with machine learning¹⁵.

Among proteomic-level detection methods, the PBP2a latex agglutination method is based on antigen-antibody specific binding, with simple operation and rapid results, having high specificity and sensitivity for MRSA. However, it cannot differentiate between different species of staphylococci, and the detection sensitivity of commercial kits has some decrease, requiring re-testing after induction with oxacillin⁸.

MALDI-TOF MS-based detection methods, such as the DOT-MGA (DNA Oligonucleotide Tag-Microarray Detection of Genomic Amplification) method, achieve optimal analytical performance at 6-8 hours of incubation, with a detection rate as high as 96.4%, and both sensitivity and specificity are 100%. But the cost is high, and the limited positions on the target plate make it difficult to simultaneously detect hundreds of samples^16,17.

Among bioinformatics analysis technologies combined with omics data for resistance prediction, whole-genome sequencing technology stands out with its high resolution and comprehensive information analysis capabilities. Combined with resistance gene analysis databases like Resfinder, it can analyze whether strains carry specific resistance genes, predict resistance potential, detect single-nucleotide polymorphisms (SNPs) and resistance-associated gene mutants, while supporting storage and reanalysis of whole-genome data¹⁸. However, this method requires operation by professionals with bioinformatics backgrounds, is rarely used in daily testing, and is commonly used for epidemiological analysis or molecular surveillance of pathogens¹⁹.

The aforementioned methodologies are either high in cost, time-consuming, or still in the research and development phase. Clinically, there is an urgent need for a solution balancing "immediacy" and "accuracy" - a novel multiplex fluorescent PCR technology fills this gap by simultaneously amplifying nuc/mecA genes in a single tube, compressing the detection cycle to 1 h. It precisely bridges the time window gap in the traditional "emergency screening - resistance typing" process, providing a more time-efficient molecular diagnostic tool for nosocomial infection prevention and control. This specific PCR assay is primarily utilized for the detection of respiratory specimens, with sputum specimens as a typical example. It exhibits a sensitivity of 500 copies/mL and high specificity, while remaining unaffected by antibacterial agents. Owing to these favorable performance characteristics, this PCR assay holds substantial application value for the diagnosis of respiratory tract infections and the surveillance of nosocomial infections caused by MRSA.

Protocol

This study was approved by the Ethics Committee of Guangdong Provincial People's Hospital (Ethics No.: KY2025-510-01). Detailed information regarding the materials used in this research (reagents, chemicals, equipment, and software) can be found in the Table of Materials.

1. Specimen collection and preservation protocol

1. Inclusion criteria: With the exception of quality control materials, identify all collected samples as positive specimens using the gold standard method (culture-based assay).
2. Human sputum collection
   1. Collect 1-3 mL of deep-coughed sputum from the lower respiratory tract of subjects using sterile sampling tubes. Seal tubes immediately and transport for analysis. Process the samples immediately for testing.
3. Specimen storage:
   1. Analyze the samples collected via the above method promptly. Short-term storage is permissible under the following conditions:
      2-8 °C: ≤7 days
      -20 °C ± 5°C: ≤6 months
      -70 °C ± 5°C: ≤24 months
      NOTE: In this study, sputum samples were not treated with preservatives (such as BHI broth + glycerol, etc.). After collection, they were stored under the conditions described above, and repeated freezing and thawing was strictly avoided. Avoid repeated freeze-thaw cycles. Maximum allowable freeze-thaw cycles: ≤3.

2. Nucleic acid extraction

NOTE: Complete all procedures within a biosafety cabinet to avoid contamination.

1. For sputum sample pretreatment, mix the sample with 4% NaOH solution in the ratio of 1:4 (sputum sample: 4% NaOH solution) to liquefy viscous sputum specimens and eliminate interfering impurities, then vortex thoroughly (30 s) and incubate at room temperature (22-25  °C) for 30 min to achieve liquefaction.
2. Preparation of nucleic acid extraction reagents
   1. Take out the pre-packaged 96-well plate from the kit. Lightly flick the 96-well plate to concentrate the reagents and magnetic beads to the bottom of the 96-well plate.
   2. Carefully tear off the aluminum foil sealing film, avoiding vibrating the 96-well plate to prevent liquid splashing out.
   3. In the solutions of Column 1 and Column 7 of the 96-well plate: First, add 20 µL Proteinase K, then add 200-300 µL sample to be extracted.
3. Automated nucleic acid extraction (Magnetic bead-based method)
   1. Place the pre-packaged 96-well plate with added samples into the corresponding slot of the automated nucleic acid extraction instrument.
   2. Follow the nucleic acid extraction process as described below.
      1. Using the magnet stick on the lab chamber, transfer the magnetic beads adsorbed with nucleic acid into different reagent holes.
      2. Use the stirring set to mix the liquid rapidly and repeatedly, so that the liquid and magnetic beads mix evenly.
      3. As the liquid and magnetic beads harmoniously combine, the automated nucleic acid extraction machine progresses through the successive stages of cell lysis, nucleic acid adsorption (70 °C, 15 min, 3 times of magnetic adsorption), washing (3 times, mix for 2 min each time, then perform magnetic adsorption twice (15 s each), and elution (2 times, mix for 5 min each time, 3 times of magnetic adsorption), eventually, it gets nucleic acid of high purity.
      4. Validate the nucleic acid concentration and purity using a spectrophotometer. The volume of the nucleic acid sample was 50 µL, with the concentration ranging from 50 to 100 ng/µL, and the A260/A280 ratio being 1.8 ± 0.2.
   3. After the program ends, directly suck the elution solution containing nucleic acid from Column 6 and Column 12 for subsequent testing. If needed, store the remaining extracted nucleic acid samples at -20 °C for long-term preservation and future use.

3. qPCR Detection of S. aureus nucleic acids and MRSA nucleic acids

1. Preparation of amplification reagents
   1. Take out the nucleic acid amplification reaction solution and nuc/mecA reaction solution from the kit (nuc gene-specific primers/probe for S. aureus, and mecA for MRSA). After thawing at 22-25  °C, fully oscillate and mix evenly, then perform instantaneous centrifugation (~6000 g, 15 s, 22-25 °C).
   2. According to the number of samples to be tested, N, prepare reagents based on the total amount of N+2 (N is the number of samples, and 2 are the negative and positive quality controls). Specific steps are as follows:
      1. Prepare the reaction system mix (16 µL nucleic acid amplification reaction solution (Buffer, dNTPs, DNA Polymerase, UDG Enzyme) + 4 µL nuc/mecA reaction solution (Primers and probes for the target detection genes nuc, mecA, and the GAPDH internal reference).
      2. Add it to an appropriately sized centrifuge tube, fully oscillate and mix evenly, and perform instantaneous centrifugation (~6000 g, 15 s, 22 °C-25 °C). Then dispense 20 µL per tube into PCR reaction tubes.
2. Add nucleic acid and quality control samples:
   1. Into the above-prepared PCR reaction tubes (20 µL), respectively, add 5 µL of nucleic acid from the tested samples, positive control, and negative control, achieving a final volume of 25 µL per tube.
   2. Tightly cap the tubes and perform instantaneous centrifugation (~6000 × g, 15 s, 22-25 °C) and then place the tubes into the PCR instrument.
3. PCR amplification:
   1. Set the cycling program as follows: Pre-denaturation: 97 °C, 90 s, 1 cycle; the following 2 steps for 42 cycles: 97 °C denaturation for 2 s and 55 °C annealing for 15 s.
   2. Set the fluorescence signal detection parameters as follows: use FAM fluorescent labeling of nuc genes; VIC fluorescent labeling of mecA genes; and CY5 fluorescent labeling of GAPDH gene as the kit's internal control. Collect data at 55 °C. Upon completion of the reaction, ensure that the data is saved for future analysis.
   3. Analyze data using qPCR-specific software (SLAN 96P):
      1. Sample information: In the well plate layout diagram of the software, label the sample types (e.g., positive control, negative control, unknown sample, internal reference gene, etc.) corresponding to the sample loading positions.
      2. Placing the reaction tubes: Open the lid of the instrument's heating module, place the sealed PCR plate/tubes steadily inside, ensure they fit closely with the heating module, and close the module lid tightly (to avoid temperature unevenness)
      3. Starting the reaction: After confirming that the program settings in the software are correct, click Start Run and record the experiment ID for future reference.
      4. Stopping the run and removing tubes: After the program finishes running, wait for the temperature of the heating module to drop below 50 °C, then open the lid to remove the PCR plate/tubes, and handle the samples properly.
4. Data analysis
   1. Before data analysis, confirm three visual outcomes: 1) Amplification curves of all samples (including internal control) show a typical S-shape, with no flat or abnormally delayed rising curves; 2) The fluorescence threshold line crosses the exponential phase of the curves, and Ct values of the internal control (e.g., GAPDH) are within ≤ 38 cycles (coefficient of variation < 5%); 3) Negative/positive controls are included throughout the entire experiment to monitor the possibility of contamination.
      1. If the experiment is out of control, recheck the entire batch. If a sample shows abnormal amplification (such as an atypical S-curve or a Ct value within the critical range), recheck that specific specimen.
   2. Call up the experimental data in the software, set the threshold and baseline, and calculate the Ct values automatically or manually, as the instrument automatically selects the baseline threshold. Set the final positive determination value at Ct ≤ 36; the reference range for the internal reference gene requires Ct ≤ 38. Confirm that their amplification curves exhibit characteristic S-shaped profiles.

Results

Detection of S. aureus and MRSA using qPCR
Based on the species-specific nuc gene of S. aureus and the mecA drug resistance gene of MRSA, we designed primers and probes labeled with distinct fluorophores for qPCR detection. All quality control results throughout qPCR experiments consistently fell within recommended thresholds, confirming the reliability of detection outcomes (Figure 1).

In th...

Discussion

Accurate and early detection of methicillin resistance is critical for the prognosis of infections caused by S. aureus. Early identification of S. aureus and MRSA can reduce the misuse of broad-spectrum antibiotics and decrease the selective pressure leading to the emergence and spread of bacterial resistance.

Traditional phenotypic detection methods, such as cefoxitin disk diffusion, require an 18-24 h cultivation period and exhibit high false-negative rates for heteroresist...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated nucleic acid extractor	DAAN	Smart 32	For DNA extraction
BSC-1500IIA2-X	BIOBASE	SEDA 20143222263	Biosafety cabinet
E-Centrifuge	WEALTEC		Centrifuge the residual liquid off the wall of the tube
Methicillin-resistant Staphylococcus aureus drug resistance gene detection kit	BOJIE	ZC-HX-38-2	Detection of methicillin-resistant Staphylococcus aureus resistance genes
Nucleic acid extraction kit	BOJIE	TQ-BG-001-96B	Extract nucleic acid
SLAN Fully automatic medical PCR analysis system	HONGSHI		Data Analysis
SLAN-96S Real-Time PCR machine	HONGSHI	SLAN-96S	Fluorescent quantitative PCR amplification
Thermo Scientific NanoDrop	Thermo Scientific	NanoDrop One	Quantitative analysis of DNA, RNA, and proteins
Ultra-low temperature freezers (DW-YL450)	MELING	SEDA 20172220091	-20 °C for storing reagents
Vortex-5	Kylin-bell		For mixing reagent