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Regardless of the introduction of broad-spectrum antibiotics, pathogenic bacteria are still the main cause of life-threatening infectious diseases in the world. Prompt detection of pathogens with the lowest concentrations (<100 cfu/mL) without time-consuming procedures, such as culture or amplification by PCR is very important in disease diagnosis and the subsequent treatment regimen.


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The detection of pathogens highly relies on the lab-based molecular techniques such as polymerase chain reaction (PCR) which quite often is misdiagnosed and delayed due to the mixture of the biological sample with numerous cells and the low concentration of the target pathogenic bacteria. This hampers the prompt therapeutic regimen for some of the acute infections requiring instant antimicrobial therapy. As an alternative to the current methods, magnetic separation (MS) techniques have been introduced to shorten the detection time of pathogenic bacteria and to increase the specificity and sensitivity of the procedure toward the target entity.

A combination of immunomagnetic separation and PCR have been used to improve the specificity and early detection of some pathogens such as Mycobacterium avium subsp. paratuberculosis (MAP) DNA in raw cow’s milk. An assay for sensitive and early detection of MAP is critical to improve the health of the cows and the dairy industry. A new assay has been developed called IMS-IS1PCR to reflect its two components: immunomagnetic separation (IMS) and IS900 PCR (IS1 PCR). Immunomagnetic separation selectively enriches the population of MAP in milk samples prior to DNA amplification and detection by PCR. The use of magnetic separation is the key component to the success of this new assay. 

PCR Technique

Among the most sensitive and specific rapid-tests for pathogen detection in biological samples and clinical specimens, PCR has been known with the highest sensitivity and specificity rate with the ability to detect specific pathogens that are difficult to culture in-vitro or need a long cultivation time.  However, PCR has some drawbacks as well, such as the susceptibility to inhibitors, contamination and experimental conditions, the sensitivity and specificity relies on target genes, primer sequences, PCR techniques, DNA extraction procedures, and PCR product detection methods.

The PCR assay in diagnosis involves several critical steps, such as DNA extraction from specimens, PCR amplification, and detection of amplicons. In particular, when specific clinical specimens, such as CSF, with only a few bacteria present are tested by PCR, each procedure must be carefully designed and performed. Since PCR is based on DNA amplification, false-positive or -negative outcomes may easily occur.

Magnetic Separation

Magnetic separation has been used as an emerging technology in biomedical science by employing magnetism for the efficient separation of biomolecules (nucleic acids, proteins, etc.), cells, cell organelles and microorganisms from the chemical/biological suspensions and samples. In MS magnetic beads (1–5 mm in diameter) or magnetic nanoparticles (<10 nm in diameter) with specific surface coating properties are synthesized to attach and isolate specific particles/entities in a mix sample. MS offers many advantages: direct isolation of target molecules from the crude sample, no restriction toward low-volume samples, non-invasive, no effect on sample materials, easy to work, functional in viscous samples, recovery of small particles (diameter approx. 0.05–1 μm) in mixed biological samples, ability for large-scale purifications, low- to high-throughput automation procedures, no shear force and no cross-contamination.

A variety of magnetic separators has been introduced to meet the demands of the sector and industry in which they are used providing the option to be used in batch and continuous mode. MS techniques has been used in the detection of various microorganisms, such as late stage malaria-infected red blood cells (iRBCs), HIV virions, E. coli, purify HIV viral product in human plasma, Listeria monocytogenes, Salmonella enterica Anaplasma and Ehrlichia and cancer cells such as early diagnosis of cancer by magnetic separation of circulating tumor cells (CTCs).

Immunomagnetic separation is necessary for early diagnosis of paratuberculosis

The previous assays for MAP were fecal culture and serum ELISA. These were time-consuming and/or not sensitive enough for preclinical MAP detection. The MAP bacteria causes a chronic inflammatory wasting disease called paratuberculosis or Johne’s disease, which is responsible for the premature death of milk cows. It is a major economic loss for the dairy industry, and can devastate a herd if not detected early because MAP is readily transmitted from mother to calf and between adult cows by environmental contamination.

Early diagnosis is critical for controlling the spread of the bacteria between cows, but early detection must be accomplished before clinical disease symptoms are apparent and before major shedding of bacteria into the environment. Detection by fecal culture is time-consuming and has a low sensitivity of 23-29%. The serum ELISA method is faster but has a lower sensitivity of 15% in the preclinical stages of infection. PCR has been used previously to detect MAP DNA in raw milk samples, but the sensitivity of this is also low at 23%. The problem with directly using milk samples is that there are a lot of PCR inhibitory substances present. The assay sensitivity can be greatly improved by selectively enriching the population of MAP in milk samples prior to PCR.

How it works: IMS-IS1PCR

By this method, antibody-coated magnetic beads bind to the MAP bacteria and are collected by magnetic separation while the PCR inhibitory substances are washed out of the sample. This added step leads to greater amplification of MAP DNA by PCR and improved detection of the bacteria at preclinical stages. The magnetic beads were coated with anti-Map antibodies and incubated with the milk samples for one hour. Then the bead conjugates were separated from solution in 10 minutes by using a magnetic rack before further characterization by IS900 PCR.

Immunomagnetic Separation improves pathogen detection

The new IMS-IS1PCR technique correctly diagnosed 80 out of 147 cows that were MAP positive (55%), which is significantly better than previous methods. Only 2 of those 147 cows were diagnosed by milk culture (1.4%), 15 by fecal culture (10%), and 20 by serum ELISA (14%). In the 118 uninfected cows there were no false positives by IMS-IS1PCR as they all tested negative for MAP. The use of immunomagnetic separation greatly improved detection of MAP in raw cow milk samples, and this work suggests that IMS could be useful in the detection of other pathogens as well.

Conclusion

Detection of specific pathogenic bacteria is based on the magnetic separation rather than on the specificity of the analytical methods which are highly specific, sensitive, and repeatable. Successful detection of pathogenic bacteria is supported by the evolution of the current magnetic affinity-based assays to replace the conventional lab-based molecular diagnostic techniques.

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Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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