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PCR, and NGS Approaches:Traditional and Molecular Methods for Microbial Detection 2025

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Clinical diagnostics as well as environmental and food safety monitoring rely on the accurate identification of microorganisms. The ability to isolate and identify viable organisms through culture-based techniques has for a long time and remains the gold standard. However, the traditional techniques are often time-consuming, have low sensitivity, and are unable to detect non-culturable or fastidious organisms. To overcome these limitations, molecular techniques such as the Polymerase Chain Reaction (PCR) and Next Generation Sequencing (NGS) have been developed and widely used. Microbial DNA
can be detected with extreme sensitivity by PCR, even for those organisms which are impossible or very difficult to culture. NGS further enables comprehensive genomic and metagenomic analyses which helps in identifying emerging pathogens and antimicrobial resistance determinants. This review aims to evaluate the three methods of detection: culture-based methods, PCR, and NGS, and analyze their working principles and strengths, weaknesses, and cases when they are most appropriate to use. There is no question that culture is important in demonstrating the presence of live pathogens and in performing
subsequent phenotypic tests. However, molecular techniques provide faster results with greater detail. In conclusion, traditional methods coupled with molecular approaches can improve the accuracy, speed, and breadth of detection of microorganisms, which can be beneficial for medical, industrial, or environmental purposes.

1. Introduction

Every sector, ranging from healthcare to food safety, requires the precise detection of microorganisms. Culture-based methods have proven their worth in the field of pathogen detection, but their sensitivity and speed more often than not compromise the end result. As with many other fields, time is of the essence. PCR and NGS are techniques designed to fulfil this need. The purpose of this paper is to analyse these methodologies so that suitable decisions can be made for optimal microbial detection.

2. Culture-Based Methods

2.1 Principles and Applications

Growing microorganisms on selective media with controlled environmental conditions is termed as culture-based methods. These enable phenotypic characterization and susceptibility of strains to antibiotics to be determined. Clinical diagnostics as well as food safety evaluation heavily depend on this method.

2.2 Advantages

  • It is cost-effective and uncomplicated.
  • Isolation of live organisms can be performed for further analysis.
  • Well-established standardized protocols exist for the procedures.

2.3 Limitations

  • Results are typically completed within 24-72 hours.
  • There are some microorganisms that cannot be cultured in a lab.
  • Sensitivity is lower in samples containing a low microbial load or in fastidious organisms, especially.

3. Polymerase Chain Reaction (PCR)

3.1 Principles and Applications

Specific sequences of DNA can be amplified to allow the detection of microorganisms with
heightened sensitivity and specificity. This can be done through PCR. It can also be done
through its variants like quantitative PCR (qPCR) which allows for measuring the microbial
load. In clinical diagnostics, environmental monitoring, and food safety, PCR is widely used.

3.2 Advantages

  • The speed of obtaining results is rapid, frequently in a matter of hours.
  • With high sensitivity and specificity, the results are accurate.
  • Organisms that cannot be cultivated may still be detected.

3.3 Limitations

  • Personnel with proper training and specialized equipment are a necessity.
  • There is a chance of contamination that can lead to inaccurate positive results.
  • Without specific methods, distinguishing dead or alive organisms is not possible.

4. Next Generation Sequencing (NGS)

Next-Generation-Sequencing-NGS
Next-Generation-Sequencing-NGS

4.1 Principles and Applications

Through sequencing the genomes of an organism or regions such as the 16S rRNA gene,
entire genomes are analysed allowing microbial communities to be studied in detail. This is useful in metagenomics, discovering pathogens, and studying the diversity of microorganisms.

4.2 Advantages

  • Detailed cultivable and unculturable organisms that make up microbial communities are provided by these insights.
  • Ability to identify novel resistance genes and pathogens.
  • High-throughput and scalable.

4.3 Limitations

  • High cost and complexity.
  • Requires advanced bioinformatics tools and expertise.
  • Longer turnaround time compared to PCR.

5. Comparative Analysis

table

Studies have shown that PCR and NGS techniques tend to be more effective at identifying
pathogens undetected by culture-based techniques, particularly in specimens with low
microbial counts or in more challenging microbes. For example, real-time PCR proved to be
more sensitive than culture methods for detecting Listeria monocytogenes in food samples. Likewise, NGS has been shown to successfully identify pathogens from clinically culture-negative specimens.

Conclusion

Culture techniques, while valuable especially when coupled with antibiotic susceptibility testing, fail in speed and sensitivity compared to molecular techniques such as PCR and NGS. As discussed in this paper, methods used should be dictated by set requirements such as turnaround time, cost, and the extent of microbial profiling needed. A combination of these methods would provide a better solution to detecting bacteria.

Soni Yadav

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Soni Yadav

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