Pathogen Detection

What is pathogen detection?

A pathogen, or infectious agent, is an organism which causes disease to its host. Pathogens include viruses, bacteria, fungi, and parasites. Examples of pathogens that cause serious disease are SARS-CoV-2, HIV, and E. Coli. Since pathogens can essentially be present in any environment on earth, they pose a risk of infecting humans in many situations. Therefore, being able to detect the presence of pathogens is important. Water supplies, food, cosmetics, and anything consumed or that poses a danger for potential pathogenic risk are routinely evaluated for pathogens. In the case of suspected pathogenic contamination, pathogen detection methods are used to assess the presence of pathogens. 

Pathogen detection is important in the diagnosis of disease. When patients present with symptoms indicating potential pathogenic infection, samples such as blood, urine, saliva, and stool are collected and then subjected to pathogenic detection assays used for diagnostic purposes.

What are the methods of pathogen examination?

Traditionally, pathogen detection has included:

  • Microscopic evaluation,
  • Culture assays,
  • Lateral flow tests,
  • Plating methods and rapid-plating solutions,
  • and ELISAs.
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Azure Cielo qPCR

Robust system that provides sensitive pathogen detection

While all of these are effective, each is laborious, requires specially trained personnel, and can take days to get results. In recent years, molecular methods, such as real-time PCR (qPCR), have become increasingly popular for pathogen detection. This is because they can offer accurate detection without as much time and resources needed. In particular, real-time PCR provides an excellent alternative to existing methods.

How qPCR is used for pathogen detection

qPCR is a widely-used method of detecting specific DNA sequences including those from pathogens. Polymerase chain reaction (PCR) uses enzymes to exponentially amplify DNA using primers targeted to a specific sequence. If fluorescent probes or dyes are used, then as the PCR process amplifies the targeted sequence in a given sample, the level of fluorescence increases with each cycle and is measured and recorded using a qPCR machine, such as the Azure Cielo.

Since PCR can amplify specific DNA fragments, it has been used in pathogen detection. Real-time PCR can be used to detect bacterial, viral, and parasitic pathogens.  Because many pathogen genomes have been sequenced, qPCR primers can be designed that are specific for groups of pathogens or even a specific pathogen itself allowing qPCR to serve as a diagnostic tool. 

For pathogen detection, time is often critical, especially in regards to patient outcomes. qPCR allows for detection, quantification and typing of pathogens much faster than traditional culturing methods.

COVID-19 pathogen detection using qPCR

When paired with an appropriate workflow, the Azure Cielo and Azure Cielo Dx have been shown to provide sensitive and robust detection of SARS-CoV-2 RNA. Test kits are available from companies such as Promega, Perkin Elmer, and TransGen.

The Cielo is designed for multiplex experiments involving up to six different targets. With the ability to scan 16 wells simultaneously, an entire 96-well plate can be scanned for all six detection channels in just 9 seconds- each sample can be evaluated for multiple pathogens in a minimal amount of time.

For accurate pathogen detection, assays need to be able to detect very low amounts of genetic material. This has been especially true with SARS-CoV-2 where early detection and diagnosis is important. qPCR instruments need to therefore be able to detect these low amounts of viral RNA. When tested, the Cielo was able to detect as little as 0.625 copies of RNA per uL of sample meaning it can be used for reliable and rapid detection of SARS-CoV-2.

For early detection and diagnosis, it is imperative that qPCR instruments are capable of measuring very small amounts of viral RNA. Researchers estimate that the limit of detection (LOD), or the lowest concentration of RNA detected with > 95% confidence, can be as little as 1 copy of viral RNA per μL.

Ways to improve upon pathogen detection

The Cielo is a qPCR system with advanced and high-performance optical technology allowing for high-quality data generation. Boasting broad-spectrum detection capability, exceptional specificity and precision, and faster run times, the Cielo consistently provides reproducible qPCR data which makes it a superior choice for use in pathogen detection.

Being able to look for multiple pathogens within one sample is very important. Having the ability to evaluate an internal control and the suspected pathogen is crucial for the accuracy and reliability of the assay. There are many cases where multiple pathogens need to be tested for at the same time. In water or food samples where a number of potential pathogens are commonly evaluated in routine tests or during a suspected contamination incident, the ability to detect multiple pathogens is key.  In the case of pathogenic diagnostics of patients, sample size is often limited and the exact pathogen causing disease may not be clear. Therefore, being able to test each sample for a variety of potential pathogens is essential.

REFERENCES

  1. Kralik, P. (2017, Feb 2). A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front. Microbiol. https://www.frontiersin.org/articles/10.3389/fmicb.2017.00108/full#h9

Additional Resources

Document TypeDescription
Application NoteUsing the Azure Cielo™ Real-Time PCR System for SARS-CoV-2 viral RNA detectionDOWNLOAD
Application NoteGoTaq Enviro Wastewater SARS-CoV-2DOWNLOAD

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