real-time pcr (qpcr), a performing method to check for the...

Real-Time PCR (qPCR),

a performing method to check

for the presence of banned

substances

Renaville R.

Progenus sa

Gembloux, Belgium

What do we know?

The product market is moving to a worldwide market

The interpretation of the Halal guidelines is not necessary the same

everywhere

For some people, Halal is associated to a brand and not to a concept

A product formulation can be simple or complex from dozen of

suppliers. How to control all of these suppliers?

Different pork DNA/proteins detection kits exist but their performances

varies widely.

Suspicions of frauds or contaminations of Halal products by pork

tissues are frequently reported

Which is the acceptable cutoff values to be claim free or not free of

pork?

Is it 0.1% or 0.01 % or 0.0001 or 0.00000 ……??

If not free, which is the percentage of

pork in the product?

How much the detection test cost?

Is the product Free or Not Free of Pork?

Can I use the test directly

at the supermarket or only laboratory

is able to do it?

Faced to this situation ( various choice of food, cosmetic or pharmaceutical

products), the consumer is completely powerless.

In the respect of the Halal guidelines, particularly concerning risk of contamination

by pork products, the consumer has 4 central questions

How to be sure of my process and the

respect of Halal/HACCP/ISO/BRC guidelines?

How to guarantee my products?

How to control my suppliers?

Is a problem (contamination, trace) is

discovered on one of mine product,

which corrective measures I will introduce

to solve the problem?

I change the supplier

I change my process

For the manufacturer, the questions are:


How to determine the cutoff values to decide

what is a fraud, a contamination or a trace?

How to guarantee to our population the highest security

of the products and the respect of Halal precepts?

What standards should be applied in our Halal guidelines

and how to control the respect of these standards?

Are we adopt only a repressive position in a case of

contamination or a constructive position by helping

the manufacturer to adopt corrective solutions?

For the authorities, the questions are:

Which method give us the highest guarantee of

enforcing these standards?

How the informations are diffused between

laboratories, between laboratories and authorities

and finaly, between authorities and the consumers?


S

S

S

ave = Yes no pork DNA is used as standard reference

minimal risks of contamination (one step test)

functional and easy

Kit is certified Halal by HCQ (NL)

imple = Yes one step ready to use

direct quantification without additionnal manipulations

ure = Yes highest sensibility (0.0001%),

highest specificity (Suidea and Vertebrate)

robustness (different forms of food, pharmaceutical

and cosmetic products)

direct quantification

For the detection of pork DNA, the Assets of the

Progenus TagPro Pig DNA Quantification kit are

To be recommended to or by authorities, a detection kit must to meet

the requirements of the 3 S theory.

Detect protein

Cheaper for single detection

Low Sensitivity (0.1% exogenous protein detected)

Risk of false positives or negatives

Risk of cross-reactivity with proteins of other species

Affected by food thermic treatment

Results not always reliable

The results is obtained in just 15

minutes

Disadvantages of this method:

• Poor Precision

• Low sensitivity (max 0.1 %)

• Low resolution

• Results are not expressed as numbers

• Not always adapted for cooking products

• Risks of false negatives/positives

+ - ? +

Near infrared spectroscopy determine inter-

species differences in the expression of myosin light

chain (MLC) isoforms to identify meat

Disadvantages of this method:

• Only on food sample

• Low sensitivity (max 0.5 – 1 %)


FP1M

FP4 Contrôle négatif

Cassoulet Contrôle

porcFP1

MFP4 Contrôle

négatif

Cassoulet Contrôle

porc

Chicken cassoulet contamined by pig DNA

Pig DNA

control

Negative

control

Disadvantages of Traditional PCR:

• Poor Precision

• Low resolution

• Non-Automated method


• Ethidium bromide for staining is not very

quantitative

• Post-PCR processing

• Risk of lab contaminations by ethidium

bromide

Measures the amount of accumulated PCR product

at the end of the PCR cycles.

This method is highly specific with

good sensitivity (0.01 %)

Advantages of Real-Time PCR:

• Increased dynamic range of detection

• No post-PCR processing

• ready to use

• highest sensitivity at the present time (0.00001%)

• highest specificity

• quantification of DNA

This method measures PCR amplification as it occurs.

This method is quantitative, because data is collected

during the exponential growth phase of PCR

The fluorescence is measured during each cycle and the

amount of fluorescence expressed is proportional

to the amount of product.

.

Item ELISA Immuno Chroma

NIR PCR qPCR qPCR Progenus solution

Target Proteins Proteins Proteins DNA DNA DNA

Sensibility 0.1 % 0,1 % 0.5% 0.01 % 0.001 % 0.00001 %

Specificity Not always Not always Pork Pork Pork Suidea

Robustness ± ±

Only food Yes Yes Yes

Expression of the results

Values Signal

Signal

Signal

Ct Value Ct Value

Quantification Standard curve

No No Semi-quantitative

Standard curve

Internal quantification marker in the

same tube

Risk of false negative/positive results

Yes Yes Yes No No No

Rapidity -2 hours 15 minutes 1 hour 4 hours 2h30-3h 2h00

• To produce an One step ready to use kit with minimal

manipulation

• Development of an internal qualibration control (vertebrate)

• No use of pork DNA to construct standard curve

• Quantification of pork DNA in the sample

by comparison with the total vertebrate DNA

in the sample

• Direct detection and quantification of pork

DNA in the sample

Our objectives in the development of a qPCR kit were:

Our strategic process to develop the kit is:

• bioinformatics analyze of the target genome

• specific probes design

• specificity, sensibility, robustness validation

• practical validation

Using our process (bioinformatics analyzes, probe design, specificity, sensibility

and robustness validation, and practical validation) , we develop not only Pork

detection kit but alsoalso other kits

All GMO detection (soon)

Chicken, turkey, ….

Campillobacter spp, E.coli spp, E-Coli H104,….

Avian influenza A/H1N1

…..

30 samples collected in different supermarkets

All samples were labelised Halal certified

qPCR method to detect the Pork DNA

The results were:

16 samples were free of pork

12 samples were contaminated (less than 0.1 %)

2 samples were used from fraud (more than 20%)

Conclusion : without high scientific method, it is difficult to certify a product.

and then, the scientific data are a valuable information for

Halal certification

Item Progenus TagPro detection kit

Provider 1 Provider 2 Provider 3 Provider 4

Sure Highest

Simple One step Ready-to-use

Needs preparation

Needs preparation

Needs preparation

One step Ready-to-use

Save IPC + Vertebrate IPC + EPC IPC + EPC

IPC + EPC

IPC + EPC

Rapidity 2 hours 2h30-3h00 2h30-3h00 2h30-3h00 2h30-3h00

Competitivity < 5 copies < 5 copies 10 copies 10 copies < 5 copies

Sensibility 0.00001 % 0.001 % 0.01 % 0.01 % 0.001 %

Specificity Suidea + Vertebrate Pig Pig + Animal ( ?) Pig + Animal (?) Pig

Quantification Direct & Immediate

Standard curve 16 points




Coverage Food, pharmaceutics,

cosmetc

Food, pharmaceutics,

cosmetc


cosmetc


cosmetc


cosmetc

Training Free / / / /

Support Free

/ / / /

ERP Free / / / /

Price Lower with quantification

Elisa versus qPCR Progenus TagPro

Detection cost

Quantification cost

Number of kits

used in

sanitary

control

>

80 % (2012) 20 % (2012)

20 % (2015) 80 % (2015)

For example: last week, FDA recommends qPCR as the standard method for

quality control in vaccine production.

(5-6 standards +

blanco + sample) x

2 tubes/point = 16

test reactives/

analyse

(1 positive, 1

negative controls +

1 samples) = 3 test

reactives/ analyse

This service issue from the qPCR test, responds to the Halal certification’s needs

in terms of:

Progenus qPCR test applied to Halal certification is not only a scientific result

but also a flexible, complete and high-quality service for the consumer,

manufacturer and authorities.

Security

our qPCR kit is able to detect and to directly quantify

the target with high specificity and sensibility

Position adopted by FDA, for example, clearly indicates that qPCR

is the method that must be recommended for control in routine in

many cases

Progenus qPCR detection test is integrated in a global service

Proof

More and more products are certified Halal. Unfortunately, daily

experiment shows that it is necessary to prove it.

Scientific control must guarantee the product but also to

detect the errors. Our qPCR kit is able to do it

Responsibility

To the consumers, producers and authorities are responsible

of the food quality and security.

our qPCR kit is efficient to control the critical points of industrial

process, logistic and distribution network.

Respect of values

qPCR analyze helps allay consumers' concerns about the correct and not

misleading information on the product label.

« Made in » with quality is important in manufacturing

In front of the trafic products, it is extremely important to be sure that the product is the

real product in the real packaging with the real brand

Our goals: Develoment of a Partneship to input a control certification of the manufacturing and packaging process and its terms of reference

Proof Respect Reponsi- bility

Security

Food of animal origine

Food additives

Preparation, processing, packaging,

transportation, storage

Additional labelling

requirements

ERP

Control, marketing and development

Communication network (laboratories, authorities, …)

CONCLUSIONS

Scientific control by using an efficient method (qPCR Progenus DNA quantification kit)

provides additional important information to certification.

It offers tailored solutions, in keeping with the special characteristics of each industry

certification organism, authorities.

It contributes to a high certification level.

It offers a competitive price for simultaneous detection and quantification of pork DNA.

It provides data that are filtered and transformed into certification, management or

business information, then forwarded to the ERP available for all partners.

27

Quality control is the real scientific tool of regulation value chains in compliance with the requirements of consumers

Mission of Progenus

The cycle at which the

amplification plot crosses this

threshold (= cycle threshold or

Ct) is proportional to the initial

amount of target sequence. Baseline

The fluorescence is measured during each cycle

and the amount of fluorescence expressed is

proportional to the amount of product.

We use the software, BioXpress developped in our laboratory

for aligment of available sequences (more than 16 109 informations in our data bank)

for identification of a sequence specific for Suidea (for example, to control of the absence

of cross reaction between our probes and donckey)

for identification of a common sequence of all knowed vertebrate species

to design specific probes for Suidea and vertebrate

Analysis of 16 million of informations

Selection of DNA

fragments what we

want and

rejection of all

false positives

DNA fragments

that could be

assimilated

Mixed meat containing 0.0001% pork (ct = 38,5 )

TagPro Sensibility

Validation by using our own electronic PCR method

of the theorical qPCR conditions (salt, temperature, cycles, …)

Species Suidea probe

Vertebrate probe

Species Suidea probe

Vertebrate probe

Pig + + Fishes - +

Wild boar + + Dairy milk - +

Warthog + + Eggs - +

Cattle - + Wheatmeal - -

Sheep - + Potato - -

Goat - + Tomato - -

Horse - + Aubergine - -

Donkey - + Mushrooms - -

Chicken - + Garlic - -

Duck - + Onions - -

Birds - + Olives - -

Dog - + Artichoke - -

Cat - + Rocket - -

The performance is dependent of the quality of input material (probes,

primers) but also of sample DNA preparation methods.

Dilution Ratio of

pork meat

TagPro Provider 1 Provider 2 Provider 3 Provider 4

1 + + + + +

10 + + + + +

100 + + + + +

1 000 + + + + +

10 000 + + + + +

100 000 + + + + +

1 000 000 + - - - -

10 000 000 + - - - -

The PIG PCR limit of detection is 5 copies of DNA.

Copy number/PCR Eff.

107 106 105 104 103 102

Operator 1

15.00 18.35 22,10 25.26 29.26 31.68

97.45

Operator 2

15.46 18.64 22,33 25.41 29.14 32.46

96.06

Operator 3

15.18 19.00 22,22 25.63 29.02 32.29

96.84

Oper.ator 4

15.47 19.12 22,26 25.71 29.06 32.00

100.50

Robustness 98.86

6 dilutions of a standard Pork DNA

4 different operators

Standard curve versus direct quantification

Mixed meat containing 0.0001% pork (ct = 38,5 ± 1) Mixed meat containing 10 % pork (ct = 22 ± 1)

Positive pig result in coconut oil certified Halal

Vertebrate

DNA IPC

control

PIG DNA

Halal certified product

From a test

to a kit

The kit contains three PCR systems:

-one for the detection of a Suidea specific gene

-one for the detection of a Vertebrate gene

-one for the detection of an internal positive control (IPC)

The three PCR systems are present in

a ready-for-use PCR mastermix allowing

the realization of the

three assays in a single reaction.

Suidea +

Vertebrate +

IPC PCR system

One reaction’s tube

per analyse

Adding 20 µl

of the mastermix

solution

The kit contains three PCR systems labeled with three different dyes in order to allow the

simultaneous quantification of the three targets.

dye

Suidea detection FAM

Vertebrate detection Vic

IPC detection Cy-5

Complex matrice Simple matrice

Automated extraction (limited raw material volume

in the sample)

IPC 5µl

Manual extraction (more raw material volume

in the sample)

+

+ 711

possible combinaisons

to produce the same biscuit

11suppliers/ingredient 7 different ingredients

More complex and

sophisticated is a

product, more

difficult is to

control

the suppliers and

the inputs

CONCLUSION

………… to a complex network

real-time pcr (qpcr), a performing method to check for the...

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