Application of Peptide Nucleic Acids in Microbial Diagnosis

Peptide nucleic acid (PNA) is a structural analog of deoxyribonucleic acid (DNA) with a neutral amide backbone, capable of specific hybridization with DNA and exhibiting high biological stability. It can sequence-specifically bind to ribosomal RNA (rRNA), with its backbone structural unit being N-(2-aminoethyl) glycine, and the base portion connected to the main backbone's amino nitrogen through a methylene carbonyl group. Its neutral backbone eliminates electrostatic repulsion compared to DNA-DNA or RNA-DNA complementary strands, thereby possessing high DNA or RNA affinity and thermal stability. Compared to traditional probe techniques, PNA probes exhibit unique advantages in microbial rapid diagnosis in fields such as food, environment, and clinical settings due to their special structure and properties.

According to the characteristics of different microorganisms and the requirements of diagnostic methods, rapid microbial diagnosis mainly employs three strategies: (1) direct detection of samples, such as fluorescence quantitative PCR; (2) short-term bacterial enrichment before detection, achieving faster results compared to traditional culture identification; (3) rapid identification after isolation and cultivation. PNA probes targeting rRNA have been successfully applied in the diagnosis using all three strategies. Based on the high specificity and sensitivity of PNA binding to nucleic acids, various detection modes have been developed, including fluorescence in situ hybridization based on microscope slides, membrane-based fluorescence in situ hybridization, chemiluminescent in situ hybridization, liquid-phase hybridization, and quenched PNA (Q-PNA) PCR techniques.

Peptide Nucleic Acid Probes

Peptide nucleic acid probes generally refer to short oligonucleotides of specific sequences consisting of 13-18 bases, shorter than DNA probes. Their neutral backbone provides higher hybridization affinity than DNA, while the specificity of base pairing is significantly improved, allowing distinction between different nucleic acid sequences through single-base mismatches. Hybridization conditions are milder compared to DNA probes, not affected by low salt concentrations. Moreover, the non-natural peptide backbone can resist degradation by nucleases and proteases, ensuring high biological stability.

• Q-PNA PCR

Q-PNA PCR is a real-time amplification detection method, using a quenched peptide nucleic acid to quench the fluorescence signal generated by a fluorescently labeled DNA primer. By adding a short tag sequence and a fluorescent label at the end of a primer, the quenched peptide nucleic acid complementary to the tag sequence brings the quencher close to the fluorescent dye. During PCR, as the primer is incorporated into the amplicon, the Q-PNA is displaced, releasing the fluorescence of the labeled primer. The specificity of Q-PNA PCR is also determined by the fidelity of the primer. Additionally, by using a mixture of primers with different labels, multiplex PCR analysis can be performed.

• Self-Reporting PNA Probes

Self-reporting PNA probes do not emit fluorescence or self-quench before binding to the target sequence. When they hybridize with the target sequence, the probe emits fluorescence. The advantage of such probes is the elimination of separation and washing steps post-hybridization. For instance, by attaching fluorescent dyes and quenchers at the ends of peptide nucleic acid molecules, the conformation of the probe in aqueous solution allows fluorescence quenching. When the probe binds to the target sequence, the fluorescence of the peptide nucleic acid dye is restored. Self-reporting PNA probes can be used for real-time PCR analysis and exhibit high sensitivity and specificity.

• PNA Blocker Probes

Although the specificity of PNA probes is significantly enhanced compared to DNA probes, their specificity may not be precise enough to distinguish sequence molecules with very close affinity to the target molecule. In such cases, unlabelled blocker probes can be used to shield non-target sequences, reducing erroneous binding of labeled probes and increasing specificity. PNA blocker probes are sometimes used concurrently with other blocking agents in molecular biology experiments. For example, in membrane-based in situ hybridization experiments, mixing with casein, albumin, fetal bovine serum, or gelatin can be used to eliminate non-specific binding of labeled probes to the membrane, enhancing specificity. Although PNA blocker probes only weakly compete with labeled probes for target binding, they significantly increase fluorescence signals. By adjusting the concentration of blocker probes and continuously optimizing hybridization schemes, optimal specificity can be achieved.

Peptide Nucleic Acid Probes in Microbial Diagnosis

• Direct Detection and Identification

Direct detection and identification of pathogenic bacteria or contaminants in samples without cultivation are the ultimate goals of rapid diagnosis. Molecular biology methods such as fluorescence in situ hybridization (FISH) and PCR can be directly used for detecting and identifying specific microorganisms, becoming essential methods in modern microbial diagnosis.

PNA-FISH directly detects microorganisms: PNA-FISH technology has emerged as a new technique for microbial detection. Group B streptococcus (GBS) is an important pathogen causing infections in newborns and the female reproductive tract. However, the false-negative rate of the currently used plate culture method can be as high as 50%. Applying PNA-FISH to detect GBS in cotton swab specimens has shown high sensitivity and specificity. Notably, this method is applicable to all serotypes of GBS (including hemolytic and non-hemolytic strains). The hybridization conditions are relatively mild, yet PNA can still penetrate bacterial cell walls while maintaining intact bacterial morphology. Compared to DNA-FISH, PNA-FISH significantly reduces detection time, possibly due to the stronger hydrophobicity of PNA compared to DNA. Furthermore, FISH technology is widely used to analyze the composition of complex microbial communities in natural environments, enabling detection of uncultured microorganisms. PNA probes targeting rRNA can provide information on microbial morphology, quantity, spatial distribution, etc.

• Identification after Cultivation

Membrane-Based PNA-FISH: Microorganisms from samples are filtered through a membrane, which is then placed on a tryptic soy agar plate for cultivation. Peroxidase-labeled PNA probes (designed for specific sequences of the 16S rRNA fragment) are hybridized in situ with the colonies on the membrane, and the light signal generated by the reaction between peroxidase and chemiluminescent substrate is captured. Based on the principle that each spot represents a colony, the number of target bacteria in the sample can be determined. Fluorescence-labeled PNA probes, such as Cy3-labeled PNA probes, can be used for hybridization with Escherichia coli 16S rRNA after cultivation and filtration, followed by PNA-FISH analysis of the membrane.

Slide-Based Solid-Phase PNA-FISH: Slide-based PNA-FISH was successfully used for detecting acid-fast positive Mycobacterium tuberculosis and Nontuberculous mycobacteria in 1999. The basic method involves preparing bacterial cultures into smears using standard methods, fixing them by flame or other chemical methods, then incubating with hybridization buffer, washing with detergent for 30 minutes, and finally observing under a fluorescence microscope. No bacterial digestion treatment is required, as PNA probes can directly penetrate the cell wall and hybridize with rRNA. Researchers confirmed experimentally that the detection limit of PNA probes for Salmonella in blood and infant formula samples can reach 1 CFU/10 mL (g), and even in cases where the target bacterium is not the dominant species among microorganisms, good sensitivity is maintained. The entire detection time is shorter than conventional PCR detection cycles. This rapid and accurate diagnostic technique not only expedites the process of determining appropriate treatment but also helps optimize control measures for infection prevention, showing great potential in clinical applications.

Liquid-Phase PNA-FISH: Building upon membrane-based and slide-based PNA-FISH technologies, researchers have developed a new method called liquid-phase PNA-FISH, suitable for fluorescence microscopy or flow cytometry. The principle involves hybridizing washed and fixed bacterial samples with a buffer containing PNA probes, followed by fluorescence detection on slides or using flow cytometry. Reports have described four types of PNA probes targeting Staphylococcus aureus, Pasteurella, Pseudomonas aeruginosa, and Salmonella, respectively, with two of them labeled with diethylaminocoumarin (DEAC), one with Cy5, and one with fluorescein. By using a mixture of these four differently labeled PNA probes for multi-fluorescent analysis of bacterial mixtures, different fluorescent filters can be selected for simultaneous identification of the four microorganisms.

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