Table 2.
Advantages and disadvantages of selected detection methods.
| Advantages | Disadvantages | |
|---|---|---|
| Conventional techniques | ||
| Selective plating | Inexpensive | Cannot culture VBNC state cells |
| Well-established | Variable specificity | |
| Can customize antibiotic makeup | Can be affected by culturing methods | |
| Immune-based | ||
| ELISA | Can perform many samples at once | Loss of sensitivity and specificity in mixed cultures |
| Several different possible techniques (direct, indirect, sandwich) | Cross-reactivity between closely related species | |
| Can change selectivity based on targeted epitopes | False positives from complex matrices | |
| Flow cytometry | Multiple parameters analyzed | Expensive, specialized equipment |
| Single cell analysis | Requires highly trained personnel to prepare, run, and analyze data | |
| High specificity | Relatively slow | |
| PCR-based | ||
| Conventional PCR | Better specificity than plating | Non-specific binding of similar DNA |
| Can be combined with other assays such as ELISA | Must be optimized | |
| Relatively simple and quick | Can only be used for presence/absence | |
| Multiplex PCR | Assay multiple species at once | Requires highly specific primers |
| Higher throughput than conventional PCR | Difficult to optimize | |
| Less costly than running multiple assays | False negatives/positives | |
| qPCR | High sensitivity | Complex matrices may include inhibitors |
| Used for rapid detection | Require highly specific primers | |
| May be multiplexed | Cannot differentiate live/dead cells | |
| EMA/PMA may be used to help distinguish dead cells | ||
| dPCR | Cheaper than qPCR | Cannot differentiate live/dead cells |
| Less vulnerable to inhibitors than qPCR | Greater chance of false positives than qPCR | |
| No calibration or internal controls required | ||
| Sequencing | ||
| 16S rRNA | Highly conserved region found in all bacteria | High cost of equipment |
| Able to distinguish species using variable regions | Relative abundance may be skewed by copy number | |
| Can be used on non-culturable bacteria | Possible species level resolution issues | |
| Whole genome sequencing | Open access of many databases | High cost of equipment |
| High discrimination | Specialized training required | |
| Can detect antimicrobial resistances and virulence genes | Varied interpretation of data | |