What is species determination? A focus on cross...

What is species determination? A focus on

cross-contamination and adulteration.

Dr Harris Steinman

FACTS

DNA Evaluation: Methods

• Protein methods

• Liquid chromatography – LC / HPLC

• Isoelectric focusing

• Immunoassays

• ELISA

• DNA methods

• DNA hybridization

• PCR

• Species

Methodology

• DNA Extraction

• DNA Evaluation

DNA Extraction

DNA Extraction

Potential problems

• Degree of cooking

• Matrix

• Homogenisation

• Sample size

• Environmental contamination

• Expertise!

DNA Evaluation

PCR

• Polymerase chain reaction (PCR) Amplification with Species-

Specific Primers

• Amplification of a specific segment, and detection

• Starting with only a very small amount of material, a DNA

segment can be multiplied by over a million-fold

• Primer: a short, single-stranded piece of DNA that anneals

(attaches) to its complementary sequence on the template. A

pair of primers will bind to either side of the target DNA segment

providing initiation sites for DNA synthesis.

DNA Evaluation

Potential problems

• results valid only if the appropriate controls have been employed

• the target sequence to be amplified

• the non-target DNA (“burden” DNA) load

Species determination

24 species-specific capture probes on each field of the LCD array

Methodology Used To Test For Meat Species

• A DNA-based LCD array is used to test for the presence of 24

animal species (17 meat and 7 poultry species).

• PCR primers used in the array target ribosomal RNA sequences

within the mitochondrial DNA of animal species.

• Primers target a small DNA fragment (~125 bp) enabling the

analysis of both raw and processed meat samples. Each field on

the LCD array contains capture probes which are specific to the

24 species being tested

• The 24-species LCD array has been thoroughly validated for its

sensitivity and species specificity

DNA Sequencing

Depends on availability of known sequences

Information present in databases (e.g. Genbank:

http://www.ncbi.nlm.nih.gov/) with a large number of

sequences from common animal species, breeds, and

genetic variations

Can be used to identify species in unknown samples

Depends on a unique DNA sequence

http://www.ncbi.nlm.nih.gov/

Factors that can give rise to variation in DNA-

based species detection results

• The sample itself

• Different methods used for DNA extraction

• PCR inhibitors

• Target DNA

• Processing and DNA degradation

• Sensitivity of the method employed (detection limits)



The sample itself

• undeclared component in a food product is seldom

uniformly or homogenously distributed throughout the

entire batch

• differences in the concentration of the contaminants at

different stages and in different products during the

production cycle.

• Homogenous vs Particulate fragments



Different methods used for DNA extraction

• Comparing the ability of five different methods to extract

DNA from fish muscle, found that DNA yields differed from

37 μg to 1246 μg, depending on the method used.

(Cawthorn 2011)

Cawthorn, DM, Steinman HA & Witthuhn RC. (2011). Comparative study of different methods for the extraction of DNA from fish species

commercially available in South Africa. Food Control, 22(2), 231-244.



PCR inhibitors

• The presence of certain compounds [originating either

from the food matrix (such as proteins, lipids, phenolic

compounds and calcium) or from chemicals used during

the DNA extraction procedure] can inhibit PCR

amplification and give rise to false negative results.



Target DNA

• Sensitivity of the method and the ability of the method to

detect target DNA influenced by the DNA target chosen for

PCR amplification.

• Mitochondrial DNA (mtDNA) targets typically used for

species detection since mtDNA is highly discriminative

and has a much higher copy number than nuclear DNA

(nDNA). Nuclear DNA only has 2 copies per cell.

• Therefore detection methods targeting mtDNA will be

much more sensitive, with much lower LODS than those

methods targeting nDNA.



Processing and DNA degradation

• Extensive processing of food products leads to a

decrease in amplifiable DNA and can influence the ability

to detect target species in a food matrix.

• During processing steps (e.g. heating, canning, acid or

enzyme hydrolysis) DNA is normally degraded into very

small fragments (often <200 base pairs (bp)) and this

needs to be taken into account when selecting a method

for species detection.



Sensitivity of the method employed (detection limits)

• The sensitivity or LOD of DNA tests with respect to target

DNA copy numbers is mainly dependent on:

o Yield (concentration) of DNA extracted with a given

DNA isolation protocol

o Number of PCR cycles employed

o Nature of starting material (raw meat or processed

meat or 'fast food’ preparations, e.g. lasagne)

Example

FACTS testing summary

Sample analysed: xxxx

FACTS Method Target species FACTS Other Lab

DNA-based Meat 4.0

LCD array kit (LOD =

0.05% to 1%)

Beef (Bos spp.) Detected Detected

Water buffalo Detected Not Detected

Chicken Detected Detected

Pork (Sus scrofa) Detected Not Detected

Example




DNA-based Meat 4.0


0.05% to 1%)





Method used to detect water buffalo was based on DNA

sequencing. The Sanger sequencing method used is not suitable

for the detection of small amounts of DNA (as would be expected

with contamination) as sequencing is typically used for species

identification, not for species detection. This is reflected by the

reported LOD of the method as 10% water buffalo.

Example




DNA-based Meat 4.0


0.05% to 1%)





Qualitative PCR Detection of pig DNA (End-point PCR,

derived from Meat Science, 44(4), 285-291)

Method designed for the detection of DNA from male pigs (boars)

only.

This method would not be capable of detecting pork in a meat

product if this was derived from a female pig (sow).

Adulteration / Contamination

Can we tell the difference between

adulteration/substitution versus

contamination?

Cross-contamination / Adulteration

Cross-contamination – depends: microbial, allergen, etc.

Transfer of species from one site/product to another.

• Direct Cross Contamination

• Indirect Cross Contamination

Adulteration / Substitution

Adulteration / Contamination

Depends on level of “contamination”

- therefore -

Can this be measured accurately?

At what percentage does it become

“adulteration”?

(substitution)

Reasons

Allergy

Religious reasons

Accurate labelling (choice)

Fair-trade

Fraud

Qualitative

Quantitative

Depends!

• Soy

• Gluten

• Meat

• Other

Qualitative

Quantitative

Ballin NZ, Vogensen FK, Karlsson AH. Species determination - Can we detect and quantify meat adulteration? Meat Sci 2009

Oct;83(2):165-74.

Meat content expressed as weight/weight (w/w). DNA and protein

analytical measurements used in quantification of species, are,

however, not easily correlated to meat content (w/w), for several

reasons.

DNA Evaluation

Where is the DNA found?

Is the DNA “amount” consistent?

DNA evaluated

Mitochondrial DNA

Nuclear DNA (Genomic DNA)

Ribosomal DNA

Mitochondrial DNA in PCR methods - low limit of detection

(LOD)

Genomic DNA - only one copy genomic DNA but many copies

of mitochondrial DNA

Mitochondrial DNA (mtDNA) targets typically used for

species detection since mtDNA is highly discriminative

and has a much higher copy number than nuclear DNA

(nDNA). Nuclear DNA only has 2 copies per cell.

Number of mitochondria DNA copies varies in different

tissues

Beef Chicken

Kangaroo

Kangaroo

Problems with DNA based methods

Differences in:

Species genome size

Tissue cell size

DNA extractability

DNA degradation

Mitochondrial distribution

Fat and water content (i.e. cell number per gram meat)

How much allowed?

1%

European Union (2003). Commission Directive 2003/126/EC on the analytical method for the determination of constituents of animal origin for

the official control of feeding stuffs. Official Journal of the European Union, L33978, 84. http://faolex.fao.org/docs/pdf/eur40447.pdf

Commission Directive 2003/126/EC of 23 December 2003 on the analytical

method for the determination of constituents of animal origin for the official

control of feedingstuffs (2) provides that official analysis of feedingstuffs with

a view to officially controlling the presence, identification and/or estimation of

the amount of constituents of animal origin in feedingstuffs is to be carried

out in accordance with that Directive. (http://ec.europa.eu/food/food/biosafety/tse_bse/docs/r09-163.pdf)

At present, the FSA was using a 1% level as "pragmatic".

How much allowed?

1%

“equivalent of at least 1%”

1 gram in 100 gram of mince/meat

10 gram in 1000 gram

1 kg in 100 kg

http://www.publications.parliament.uk/pa/cm201213/cmselect/cmenvfru/1085/1085.pdf

“to explore the limits of tolerance of trace contamination”


Health?

Halaal?

Manufacturing?

The project will look at unavoidable carry over between processing of

different types of minced meat through the same equipment or prepared

sequentially on the same production lines according to good manufacturing

practice.

Simultaneously, FSA is undertaking research using established consumer

forum methodology to explore consumer acceptability of trace level

carry-over.


In addition, Government is working with LGC to carry out research to

fully validate limits of detection for methods to detect DNA, used by

Public Analysts to support accurate interpretation of meat speciation

survey results as a whole in a standardised way.


Taken together, this research on industry achievability and consumer

acceptability will inform a decision on an appropriate future threshold for

trace levels under good manufacturing practice in food production and

processing.

. . . research using DNA testing to establish what level of cross

contamination between meat species occurs in industrial practice on

production lines with multi-species plants where the most stringent and

rigorous cleaning regimes are in place.

. . . . will gather evidence to help define a threshold trace level as good

hygiene practice in food production and processing.


Until that work is completed, it seems premature to consider the

option of regulating to require separate production lines for

different meat products.


Disclaimer

Harris Steinman

Carine Davies Registered Dietician

Karen Horsburgh

Registered Dietician

Donna Cawthorn Food Scientist (Ph.D.)

Shafeeqah Allie Administration

Corrine Hugo Food Scientist

Debora v.d. Merwe Food Scientist (Ph.D.)

Comaine van Zyl Food Scientist

www.factssa.com [email protected]

What is species determination? A focus on cross...

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