Limitations of infection detection methods used today

  • General comparison
  • Culturing
  • Rapid test kids
  • PCR
  • MALDI-TOF

General comparison

The methods widely applied for bacteria detection in veterinary practice are shown in Table 1. However, these methods are either unreliable (quick test), expensive (MALDI-TOF) or slow (culturing, PCR).

Table 2. Methods currently used for bacteria detection in veterinary practice.

Method Principle of the identification Advantage Drawback
Classical culturing in Petri dish[1] Morphology Standard, well-known, easy Time consuming (12-48 h), human factor in the measurement, sterile laboratory environment, price: 10-40 € per sample (depending on the bacteria and country of location).
Rapid test Colour change, due to pH change Relatively rapid, small, easy to handle, Not reliable (less than 60%), relatively expensive (10 €), single use only
Polymerase Chain Reaction (PCR) [2] DNS detection Accurate, small size, database is required. Relatively slow (12-24 h), expensive (50 €), dead bacteria are also detected (complicating factor)

MALDI-TOF/MS [3]

 

Structural analysis of proteins and their fragments a Rapid (1-2 hours), accurate Expensive equipment (300-500 thousand euros investment), high vacuum, high maintenance costs, difficult sampling preparation. Cost per sample is about 100 €

[1] In Hungary, these methods are standardized: MSZ–364/4–86, MSZ–- 3640/18–1979, MSZ–3640/12–197

[2] WO 2006/085948

[3] US 6177266

Culturing

Culturing of different bacteria in one petri dish

The most widely applied method is microbiological culturing. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the best temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius for cultures from humans or animals, or lower for environmental cultures). Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. The identification of the bacteria is made upon visual identification, which makes the method unreliable. Determination of several bacteria at the same time meets also difficulties. But the largest drawback so far is the waiting time of minimum 12 hours, typically 48-72 hours. The test requires human intervention, and the cost is between 10 and 50 €/test, depending on the country (high labour cost).

 

Rapid test kid

Rapid test kid

Rapid test kids are usually based on specific reaction or reagent. Using this method only the dedicated bacteria can be detected, so the user must have a clear preconception about the possible disease. No rapid tests are available for all kinds of bacteria. Its major advantage is the short response time (few minutes), and the drawback is its low reliability. Nevertheless, its price is quite expensive, can reach 10 €/test.

 

 

 

 PCR

The principle of the PCR method

Polymerase Chain Reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies
of a particular DNA sequence. PCR permits diagnosis diseases. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses. PCR also permits identification of non-cultivatable or slow-growing microorganism
s. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes[4]. The main advantage of PCR is its high accuracy, small size.  Its drawback is that the response time is relatively slow (12-24 h), and also expensive (50 €). As a complicated factor dead bacteria can also be detected.

 

MALDI-TOF

MALDI-TOF spectrometer

Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) is a special type of mass spectrometry that can be used for rapid and reliable detection and identification of bacterial species[5].  MALDI
TOF are usually applied in diagnostic of humans and identification of different organism. In MALDI-TOF the high molecular weight DNA or protein species are identified. MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them[6]. Its major advantage is its high speed (1-2 hours), high accuracy. On the other hand, the equipment is very expensive equipment (300-500 thousand euros), requires high vacuum, high maintenance costs, difficult sampling preparation. The cost of one measurement can be up to 100 €.

[4] Cai, H; Caswell J.L.; Prescott J.F. Veterinary Pathology 51 (2): 341–350.

[5] Silpak Biswas, Jean-Marc Rolain, Journal of Microbiological Methods Volume 92, Issue 1, January 2013, Pages 14–24.

[6]  Karas, Michael; Krger, Ralf (2003). Chemical Reviews 103 (2): 427440.

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