Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Pathogens are bacteria, microbes or viruses that invade the cells or tissues of an organism and cause damage. Paramount to defending ourselves against pathogens is our ability to detect them. In fields like clinical diagnostics and research, drug discovery and development, forensics, biodefense and food safety, pathogen detection is a crucial tool. When researchers can detect pathogens from cells or tissues reliably, they’re better able to study responses to infection or responses to an experimental treatment against the pathogen. Recently, older culture-based methods have been outpaced by newer, nucleic acid-based techniques, which offer greater convenience, speed, sensitivity and specificity. The following are the most common types of nucleic acid-based methods currently used to detect these often deadly agents.
Detection by sequencing
New sequencing methods have an advantage over other means of pathogen detection, which often require you to know the sequence of the pathogen ahead of time. If you don’t know the sequence—such as with an outbreak of a new strain, spontaneous mutations or simply very rare pathogens—detection by sequencing is a beneficial alternative. It involves sequencing infected cells and comparing the sequences to a reference library of pathogen sequences for identification. This option is increasingly successful for identifying pathogens because of the ever-increasing number of sequences that are publicly available. Another benefit of sequencing methods is that all pathogens present in the infected cell samples show up in the results—not just the most numerous pathogens or the fastest-growing pathogens (toward which the older, culture-based methods could be skewed). Sequencing methods of detection are also a good choice when you need to process samples quickly for rapid turnaround.
The reason the newest sequencing detection methods are so fast is that they take advantage of next-generation sequencing methods. Some protocols use DNA as a starting sample, and others use RNA, depending on which fits the researcher’s system best. Using small RNAs (20 to 50 nucleotides) can have the advantage of larger pathogen-to-host ratios compared with using DNA (which is usually more abundant in the host); also, bacteria and viruses are more likely to have degraded or fragmented RNA than the host, allowing for greater enrichment of pathogen short RNA. [1]
For use in clinical settings where results are needed quickly, sequencing methods are also best. Swab samples from the insides of patients’ cheeks can be analyzed using next-generation sequencing, for example, in cases of new viral outbreaks, in which the sequence of the virus may be unknown. [2] Also, other methods of pathogen detection would be unsuccessful in cases in which a previously characterized virus had mutated in primer-binding regions. Detection by sequencing, however, could still be successful—and in addition, it might identify the mutation.
Detection by qPCR
Although they do require previous sequence knowledge, pathogen-detection methods that rely on real-time or quantitative PCR (qPCR) are also sensitive and convenient. This method works on the principle that each type of pathogen has a unique “signature” of DNA or RNA that sets it apart from others. After you have a reliable nucleic acid signature, you can test for your pathogen of choice quickly and reliably, often within an hour or less. Another useful aspect of this method is its ability to quantify the targets of the test, for example, to assess the level of particular bacterial species in the test sample.
Testing kits that use this method are especially valuable in clinical settings and food-safety testing. For example, physicians can test for the presence of suspected bacterial strains quickly, while the patient remains on-site, thereby sending the patient home with the correct antibiotic. In food-safety situations, manufacturers offer a range of food-safety testing kits for all pathogens known to contaminate food sources, such as commonly found Salmonella or E. coli O157 strains.
Detection by microarray
Pathogen detection also can be accomplished using microarray-based techniques. In this method, you apply pre-amplified sample DNA to a microarray consisting of thousands of short, single-stranded DNA oligonucleotide probes from the targets of interest. This method is helpful when you want to screen one sample for a wide range of possible pathogens quickly and in parallel. For example, with one microarray you can test for pathogens such as B. anthracis, Vaccinia, monkeypox, F. tularensis (tularemia), Y. pestis (the plague), ebola, Marburg virus and Dengue 1-4. These arrays are usually readable by standard fluorescence microarray scanners. This method offers specific answers with high throughput. However, as mentioned above, if you need to identify a pathogen, yet you don’t have a well-founded suspicion of its identity, then the sequencing methods discussed above may be more appropriate. For increased confidence, it may also be a good idea to verify any positive microarray results with a sequencing- or qPCR-based method of detection.
References
[1] Isakov, O, Modai, S, Shomron, N, “Pathogen detection using short-RNA deep sequencing subtraction and assembly,” Bioinformatics, 27(15):2027-30, 2011.
[2] Yongfeng, H, Fan, Y, Jie, D, Jian, Y, Ting, Z, Lilian, S, Jin, Q, “Direct pathogen detection from swab samples using a new high-throughput sequencing technology,” Clinical Microbiology and Infection, 17(2):241-4, 2011.