3_DETECTION OF MICROORGANISMS IN FOODS: METHODS AND TECHNIQUES

foodborne illness

Microbes can contaminate foods and cause

• Culture-based methods

• Nucleic acid-based methods

• Phage-based methods

Detection of microbes in food can be done through:

Viability of cells

is the ability of bacterial cell to replicate in liquid culture media or to produce a visible colony on solid culture media

• Sub-lethality injured

• Viable-but-non-culturable (VNBC) or ‘persister’

• Dormant

Microorganisms in food may exist in three metabolic or physiological states:

Injured cells (sub-lethally or severely)

• Damage to essential cell structures and cell functions

• Limited ability to grow on selective media; presence solely and predominantly demonstrated on non-selective media

Viable but non-culturable (VNBC) “Persister”

• Low but detectable metabolic activity (genes expressed and proteins produced)

• Membrane integrity maintained

• Formation of colonies on solid culture media inhibited

Dormant

• Shutdown of the metabolism

• Negligible metabolic activity not detectable by viability assays

• ·       totally inactive of cell, but not dead, but can be viable when nutrients are available -> due to being exposed to osmotic stress/lack of nutrients

• Pre-enrichment

• Selective enrichment

• Plating on selective media

• Biochemical or serological tests

Series of steps is required before definitive identification can be confirmed:

• Sample collection and Processing

• Enrichment

• Serial Dilution

• Plating

• Isolation

• Biochemical, Serological, Drug Resistance Test

Process of culture:

nucleic acid-based and bacteriophage-based

Two methods for foodborne pathogen detection:

Viability PCR/qPCR

Culture-independent method:

• Pre-incubation of test sample with PMA or EMA dyes, which penetrate into bacteria with compromised cell membranes and bind genomic DNA, making it non-amplifiable.

• Gives PCR the capability to differentiate viable and dead cells more quickly than culture. qPCR provides quantitative results.

• Dead/inactivated bacterial cells do not always have compromised cell membranes, so false positives may result.

Reverse-transcriptase qPCR (RT-qPCR)

Culture-independent method:

• Bacterial transcripts are sensitive to degradation by intra- and extra-cellular RNases, so mRNA levels should rapidly decline after cell death. Thus, detectable mRNA would be limited to the viable and active cells within a sample.

• Quick compared to culture, but additional cDNA generation step makes it longer test than viability PCR/qPCR.

• Not all studies have demonstrated that mRNA is short-lived, so false positive results may occur. RT-qPCR viability assess- ment validated for longer (>200 bp) transcription products, but not neces- sarily short qPCR products.

Nucleic acid-based Methods

Operate by detecting specific DNA or RNA sequences of the target pathogenic organisms.

• Polymerase chain reaction PCR)

• Rapid, specific, and sensitive

• Do not provide indication about the viability of detected cells

• Detection of mRNA is considered better indicator cell viability than DNA, since its present metabolically active cells

• e.g., qPCR and Loop-mediated isothermal amplification (LAMP), Reverse trasncription-PCR, RT-qPCR

Phage amplification (Plaque) assay

Culture-independent method:

• Phages only replicate within viable cells and ultimately lyse these cells to release progeny phages within an agar lawn to form plaques (zones of clearing).

• A 24-h test, producing count- able plaques giving a quantitative result.

• Not suited as a high-throughput test. Laborious, multi-step test which requires cooled molten agar.

• Virucidal step is key step, otherwise false positive results may be obtained.

Phage amplification + qPCR

Culture-independent method:

• As above, but cell lysis occurs in liquid suspension, releasing progeny phages and host DNA, which can both be detected and quantified by qPCR.

• Rapid, one-day test, with op- tion to detect released phages or the host DNA by qPCR to demonstrate that lysis has occurred. Only vi- able cells lyse. Potentially a quantitative assay.

• Important that DNA is released into as small a volume as possible to maximize detection. sensitivity, otherwise DNA precipitation and column extraction may be necessary.

Phage amplification + immunoassay

Culture-independent method:

• Phage amplification proceeds until cell lysis in liquid suspension, releasing progeny phages which can be detected by ELISA or immunochromatographic test

• Rapid, one-day test similar to when qPCR is used. Only viable cells lyse. Potentially a quantitative assay.

• Analytical sensitivity more limited compared to qPCR detection after phage amplification.

Phage amplification + enzyme assay

Culture-independent method:

• Phage amplification proceeds until viable cells burst to release intracellular components such as ATP or B-galactosidase, which are measured by enzyme assay.

• Rapid, one-day test similar to when qPCR or immunoassay are used. Only viable cells lyse. Potentially a quantitative assay.

• May require genetically engineered phages. Not many food testing applications to date.

Denaturation, Annealing, Extension

PCR Process

Phages

• are highly specific and has natural affinity to the host cells.

• They can only replicate inside living cells, thus ___-based methods can test to demonstrate cell viability.

Virucide

• is applied to kill all the exogenous phages

• crucial physical treatment, applied to kill all other viruses on the outside

soft agar

Samples are plated with ___and an indicator bacterium

Xylose Lysine Deoxycholate agar (XLD agar)

·       differential agar for Shigella and Salmonella in enteric culture

Spread plate

agar first, specimen after (0.1 ml) ->colonies growing above the surface

Pour plate

specimen first, (1ml), pour medium -> colonies growing above the surface and subsurface

qPCR

getting the actual quantity of the genes

mRNA PCR

better to test than DNA because better to test than DNA because

Taq Polymerase

from Thermus aquaticus

Indicator bacterium

once infected by the burst cell, it will indicate clear zone

above the surface

In spread plate, colonies grow ____

above and on the subsurface

In pour plate, colonies grow ____