Bacteriophage Based Bioluminescence Biosensor For Pathogen Detection In Food

N.A.G. Sandaruwan

Why detection of micro organisms in food is important?

“2.2 million people die annually due to food and water borne illnesses, and 1.9 millions among them are children” -WHO

Conventional methods used for this are microbiological and biochemical analysis, ELISA, PCR etc.

They are overly time consuming, not amendable to on site detection and need a extensive training.

Biosensors have been identified as attractive alternatives to the existing conventional pathogen detection methods.

What is a Biosensor?

A biosensor is an analytical device, used for the detection of an analyte, that combines a biological component with a physicochemical detector.

Classification of Biosensors

Bioluminescence

History of Bioluminescence Technique

In 1667 Robert Boyle, English chemist discovered bioluminescence.

He observed few spot of bluish-green light on a chicken carcasses which was purchased the week prior.

Dubois confirmed oxygen dependency and demonstrated the necessity of an enzyme (luciferase) and a luciferase substrate for light emission.

BioluminescenceNeed luciferase enzyme.Substrate is catalytic oxidated by

the enzyme to produce light in the presence of molecular oxygen.

FMNH2 + RCHO + O2 FMN + RCOOH + H2O

+ Light

Bioluminescence in a microorganism

Membrane synthesis

Myristol ACP

Myristic acid

Myristic Aldehyde

Light (490nm)

FMNH2

Luciferase

Luciferase

Genetic Background

Luciferase can be found in bacteria ( lux ) and eukaryotes ( luc ).

It’s encoded by lux A and B genes.

In addition light generation requires lux C,D and E genes.

How to use a Bacteriophage?

Select a specific bacteriophage

Change the DNA component

Introduce to the medium

Multification

Reporter phage with lux gene

Introducing to the

sample

Contamination with

targeted MO

Reporter phage attack

to MO

Inject lux gene

Luciferase enzyme is produced

Light emmission

Industrial applications

A511::lux AB recombinant phage is used to identify Listeria bacterium in Spikked ricotta cheese, chocolate pudding etc.

A511::lux AB is used to check the quality of minced meat and soft cheese.

P22::lux AB is used to identify Salmonella Spp.

( ex:- in eggs )

Advantages of bacteriophage based biosensers

Very sensitive.Less time consumingMinimal sample preparation.Specificity of detection can distinguish between live

and dead MO s.

Limitations

Presence of specific phage inhibition.

Antiviral bacterial immunity system.

Thank you!