A Comparison of Molecular Methods for Microbial Testing
Molecular methods have revolutionized microbial testing, offering enhanced sensitivity, specificity, and speed compared to traditional culture-based approaches. However, variations between methods exist within molecular testing in terms of cost, time efficiency, specificity, quantification capabilities, and flexibility in targeting multiple pathogens.
Nucleic acid-based methods
Traditional PCR
Traditional PCR is the foundation upon which many other molecular techniques are built. It involves cycles of denaturation, annealing, and extension to exponentially amplify a target DNA region, making it easier to detect and analyze.
First, the double-stranded DNA is heated to a high temperature (typically around 94-98°C), causing the DNA strands to separate, or denature, into single strands, providing access to the target sequence. Then, the reaction mixture is cooled to a temperature (typically 50-65°C) that allows specific DNA primers to bind to complementary sequences at the target region. The reaction temperature is then raised to the optimal temperature for the DNA polymerase enzyme (usually around 72°C). DNA polymerase synthesizes a new DNA strand complementary to the target sequence, using the primers as starting points.
This cycle repeats around 20-40 times, with each cycle doubling the amount of DNA to exponentially amplify the target sequence. The DNA products are then analyzed post-reaction, often using gel electrophoresis, which visualizes the presence and size of the amplified DNA fragments as bands on the gel.
While it is relatively cost-effective and straightforward, traditional PCR lacks quantification capabilities and can be prone to non-specific amplification, leading to false positives. Furthermore, PCR can take several hours to complete a run. However, it remains a valuable tool for initial screening or when precise quantification is not necessary.
qPCR (Quantitative PCR)
qPCR involves the amplification of a specific DNA sequence using primers and a heat-stable DNA polymerase. During the reaction, the amount of amplified DNA is monitored in real-time using fluorescent dyes or probes, allowing for quantification of the initial DNA template. The cycle threshold (Ct) value, representing the PCR cycle at which the fluorescence signal reaches a predefined threshold, is used to quantify the amount of the target DNA. As the PCR reaction progresses, the fluorescence intensity increases proportionally to the amount of amplified DNA, and a standard curve is used to quantify the DNA.
qPCR offers the advantage of quantifying the amount of target DNA present in a sample, allowing for highly specific detection of low-level targets. qPCR also provides results in real-time or with a short turnaround time, making it suitable for high-throughput applications. Additionally, it is possible to detect several sequences in a single reaction by using probes that carry different reporter dyes. However, qPCR assays can be expensive due to the need for specialized equipment and reagents.
ddPCR (Digital Droplet PCR)
Similar to qPCR, ddPCR also utilizes DNA polymerase to amplify a target DNA sequence in a standard qPCR assay. However, ddPCR partitions the whole qPCR reaction into thousands of individual droplets, each containing a single DNA molecule. The PCR amplification occurs within these isolated droplets, and each droplet is independently analyzed for the presence or absence of the target DNA. The end-point fluorescence measurement of each droplet is then used to determine absolute target quantification without the need for standard curves.
ddPCR represents a significant advancement in molecular testing, offering absolute quantification of target DNA. This technique provides more precise and reproducible data than traditional qPCR, especially when PCR inhibition is present, allowing it to detect rare targets present at very low concentrations. While ddPCR has performed better than qPCR at detecting low concentrations, qPCR is more reliable in detecting higher concentrations. Furthemore, the initial setup costs for ddPCR are high due to specialized equipment and proprietary reagents, and multiplexing is generally more limited compared to other PCR methods.
LAMP (Loop-Mediated Isothermal Amplification)
LAMP is used to amplify DNA under isothermal conditions, meaning it can operate at a constant temperature without the need for a thermal cycler. The reaction typically proceeds at a temperature between 60°C and 65°C, making it suitable for field-based or point-of-care applications where access to sophisticated equipment is limited.
The LAMP reaction begins with the denaturation of the target DNA at the constant reaction temperature. The primers form loops at their complementary sequences on the target DNA, initiating the amplification process. Similar to PCR, LAMP also uses DNA polymerase, which displaces existing DNA strands and synthesizes new DNA strands simultaneously. As a result, multiple copies of the target DNA are amplified with each cycle, leading to exponential amplification. The amplified products of the LAMP reaction can be detected using various methods, including turbidity measurement, visual inspection, or fluorescence-based detection.
LAMP is generally cost-effective, as it operates under isothermal conditions and requires fewer equipment and reagents compared to PCR. Furthermore, LAMP provides rapid results within 30 minutes to a few hours, as it does not require thermal cycling. Although LAMP tends to have a lower limit of detection than traditional PCR, meaning it can detect lower concentrations of DNA in a sample, qPCR tends to outperform LAMP in sensitivity and specificity metrics.
DNA microarray
Microarrays are chips containing thousands of known DNA sequences. The target DNA or RNA from a sample is labeled with a fluorescent dye and allowed to hybridize with these sequences. The intensity of fluorescence at each spot on the microarray corresponds to the abundance of the target sequence, providing information about gene expression levels or identifying specific DNA sequences in the sample. DNA microarrays enable the detection of multiple targets in a single experiment, which makes it suitable for the analysis of massive targets.
Microarrays have limited sensitivity for low-abundance transcripts compared to newer sequencing technologies. Cross-hybridization with non-target sequences is also a potential concern, interfering with specificity. Microarrays also tend to be expensive, with high costs associated with chips, instrumentation, and specialized reagents.
Sequencing-based methods
Next-generation sequencing (NGS)
NGS, also known as high-throughput sequencing, encompasses various platforms and technologies that enable the parallel sequencing of millions to billions of DNA fragments simultaneously. NGS platforms, such as Illumina and Roche 454, utilize different sequencing chemistries and approaches but generally involve determining the precise order of nucleotides within a DNA or RNA molecule. By sequencing DNA, NGS allows researchers to identify and characterize the diversity of microorganisms present in a sample. Additionally, NGS can be applied to whole-genome sequencing of microbial isolates for genomic analysis, outbreak investigations, and evolutionary studies.
While NGS excels at generating large numbers of reads across the genome while minimizing costs through parallel processing, it requires DNA amplification, which may introduce random errors during DNA synthesis. As DNA continues to be amplified, these random errors progressively lead to a loss of synchronization across DNA strands, resulting in declining signal quality as read lengths increase. Consequently, long DNA molecules must be fragmented into smaller segments to maintain the read quality, presenting a significant limitation with NGS. Furthermore, NGS workflows often involve library preparation, sequencing, and data analysis steps, which can take several days to weeks depending on the depth of sequencing and complexity of the analysis pipeline.
Nanopore sequencing
To overcome this limitation, Third-Generation Sequencing (TGS) technologies emerged, producing longer reads by directly sequencing single DNA molecules. For example, nanopore sequencing enables the sequencing of DNA/RNA without PCR amplification or chemical labeling. Nanopore sequencing utilizes nanopores, nanometer-sized protein pores embedded in a synthetic membrane. When a DNA or RNA molecule passes through a nanopore, it causes disruptions in an ionic current, which can be measured and used to infer the sequence of nucleotides.
Nanopore sequencing has great potential in providing relatively low-cost genotyping, real-time sequencing capabilities, and long read lengths. However, nanopore sequencing is limited by higher error rates compared to NGS, with error rates ranging from 5% to 15%. Nevertheless, recent improvements in nanopore chemistry and computational tools have led to increased accuracy, with reported accuracies reaching up to 98.3%. Despite improvements, nanopore sequencing may still have higher error rates compared to NGS.
Immunology-Based Methods
ELISA (Enzyme-Linked Immunosorbent Assay)
ELISA utilizes the high specificity of antigen-antibody interactions to detect and quantify the presence of specific proteins in a sample. Instead of identifying microbes through their DNA, ELISA and other immunology-based methods target proteins and other biomarkers present in specific microbes. ELISA uses specific fluorescently labeled antibodies to capture target antigens/proteins in the sample. Then, the intensity of the signal is measured using a spectrophotometer to quantify the amount of the target molecule.
Although ELISA methods can specifically detect targeted bacteria and their toxins, as well as be multiplexed for multiple samples, they are still limited by false-negative results and cross-reactions with similar antigens. Furthermore, immunological methods usually require pre-enrichment to expose surface antigens, which leads to extended detection time.
Conclusion
Each molecular method for microbial testing has its unique strengths and limitations, making them suitable for different applications based on requirements for cost, speed, specificity, quantification, and multiplexing. As technology continues to evolve, further innovations in molecular methods will emerge, offering even greater capabilities for microbial detection and quantification in a variety of applications.
References
Zhang S, Li X, Wu J, Coin L, O’Brien J, Hai F, Jiang G. Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis. Water. 2021; 13(24):3551. https://doi.org/10.3390/w13243551
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