final optimisation and evaluation of dna based methods for ... - Defra, UK

FINAL OPTIMISATION AND EVALUATION OF DNA BASED METHODS FOR THE AUTHENTICATION AND QUANTIFICATION OF MEAT SPECIES

H. Hird, G. Saunders, B. Popping, S. Garrett and G. Wiseman

Central Science Laboratory Sand Hutton York YO41 1LZ

FINAL OPTIMISATION AND EVALUATION OF DNA BASED METHODS FOR THE AUTHENTICATION AND QUANTIFICATION OF MEAT SPECIES

DRAFT FINAL REPORT

Authors

H. Hird, G. Saunders, B. Popping, S. Garrett and G. Wiseman

Date

16/03/05 Food Standards Agency Aviation House 125 Kingsway London WC2B 6NH

Sponsor

Sponsor’s Project No.

Q01084

FD report number.

FD04/40

Principal Workers

H. Hird, J. Chisholm, G. Saunders, B. Popping, S. Garrett and G. Wiseman

Distribution List for draft report

Mark Woolfe, FSA

Central Science Laboratory Sand Hutton York YO411LZ Tel: 01904 462585 Fax: 01904462111

2

CONTENTS Section

Page

ABBREVIATIONS

4

EXECUTIVE SUMMARY

5

INTRODUCTION

7

RESULTS AND DISCUSSION

8

Validation and comparison of qualitative real-time PCR assays

8

Comparison of extraction protocols for high quality DNA from complex matrices.

17

Proficiency trial of qualitative real-time PCR assays

29

Comparison of approaches for quantification

33

BIBLIOGRAPHY APPENDICES Appendix 1

47 Materials and methods

48

Appendix 2

DNA extraction methods

63

Appendix 3

Proficiency trial method for CSL pork and chicken Taqman assays

77

Appendix 4

Food Standards Agency Standard Operating Procedures

81

3

ABBREVIATIONS +ve

Positive

bp

Base pairs

CCFRA

Campden and Chorleywood Food Research Association

CSL

Central Science Laboratory

CT

Threshold cycle

CTAB

Cetytrimethylammonium bromide

DNA

Deoxyribonucleic acid

dNTP

2’-deoxynucleoside 5’-triphosphates

E

PCR amplification efficiency

EB

Extraction blank

EDTA

Ethylenediaminetetraacetic acid

FAM

carboxy-fluorescein

FSA

Food Standards Agency

GMO

Genetically modified organism

IPC

Internal positive control

LOD

Limit of detection

MAFF

Ministry of Agriculture, Fisheries and Food

MBGW

Molecular biology grade water

mCyt b

Mitochondrial cytochrome b gene

MGB

Minor groove binder

NCBI

National Center for Biotechnology Information

NTC

No template control

PCR

Polymerase chain reaction

RPM

Revolutions per minute

TAMRA

carboxy-tetremethylrhodamine

TBE

Tris-Borate-EDTA buffer

TE

Tris-EDTA buffer

Tris

Tris(hydroxymethyl)aminomethane

R&D

Research and development

RHMT

RHM Technology Ltd

SOP

Standard operating procedure

VLA

Veterinary Laboratories Agency

WC

Water control

w/w

Weight for weight

4

Executive summary

EXECUTIVE SUMMARY The Food Labelling and Standards Division of the FSA (formally of MAFF) funded 4 laboratories to design and optimise DNA based species-specific assays for turkey, chicken, pork, beef and lamb. Another laboratory was funded to develop methods to identify meat adulteration in vegetarian foods. The application of the developed assays and their technological transfer to enforcement and other analytical laboratories, has yet to be fully realised. The objective of this study was to validate these assays and produce a set of protocols which would provide comprehensive guidelines for the analysis of meat species in commercial meat products, and be suitable for enforcement of product labelling.

These protocols were to encompass the DNA extraction technique, the

species-specific assay conditions and quality control criteria for each assay. The project was divided into two strands which ran concurrently: •

The optimisation and comparison of qualitative species-specific real-time PCR assays

•

The comparison of different approaches to quantify species using real-time PCR assays.

The first strand of the project identified the most appropriate DNA extraction protocol suitable for the purification of high quality DNA, (in the absence of inhibitors), from raw and canned meats, including samples made from mixed species that also contained fat or rusk. It was found that a single DNA extraction protocol was suitable for all meat matrices tested:

the CTAB, proteinase K, wizard protocol and

additionally, sensitive real-time PCR assays for chicken and porcine meats were also identified. The final work for this strand of the project involved a proficiency trial of the CTAB, proteinase K, Wizard DNA extraction protocol, coupled to the porcine and chicken real-time PCR assay, used by each of the contractors to analyse a set of sausage samples. Additionally, contractors used in-house real-time PCR assays for chicken, pork, beef, lamb and turkey on the DNA extracted from the samples.

Upon

comparison of the results it was found that all the real-time PCR assays using both mitochondrial and nuclear DNA targets, previously developed by the contractors,

5

Executive summary were able to correctly identify the species present within a sample, with no false positive results. This strand of the project was completed with the provision of a comprehensive set of Standard Operating Procedures for the identification of meat species in complex food products using real-time PCR-based methods. The second strand of the project involved an investigation into different approaches to quantitate the amount of meat of a given species present in a commercial sample. This work focused on the identification of the most appropriate calibrants for quantification, and investigated the use of single and mixed meat samples, dilutions of DNA and plasmid standards. Real-time PCR assays detecting both multi-copy mitochondrial targets and single copy nuclear targets were also compared. The results of this work showed promising trends which could be used for meat quantification, however none of the calibration methods proved robust enough to be considered suitable for enforcement of product labelling. In conclusion, the project allowed the successful identification of a DNA extraction protocol and real-time PCR assays for the qualitative determination of common species present in complex foods, and which are suitable to assist public analysts and enforcement laboratories. Also, a set of standard operating procedures for these methods has been provided to the Food Standards Agency (Appendix 4).

6

Introduction

Introduction Consumers demand choice in their diets and decisions on diet can be made due to health, religious or lifestyle reasons. For example, consumers frequently exercise choice with regard to food containing genetically modified organisms (GMOs), types of meat or meat free and organically grown products. Such choices are often made solely on the basis of product labelling and certain choices can be associated with premium prices. In order to support and protect consumer choice, strict labelling regulations need to be adhered to and misdescription by the food producer or vendor needs to be minimised. Acts of purposeful fraud intending to mislead the consumer can lead to prosecution, therefore robust and forensically executed analytical tests are required to support the legislation. A recent change in the Food Labelling Regulations means that food products require a quantitative declaration (QUID) for ingredients mentioned in the name of the food. Therefore, food items where a type of meat is specified in the name of the food will have to stipulate the amount of that meat in the product. This is in contrast to the total minimum meat content required under previous regulations. This change in labelling places new requirements on analytical techniques to support the legislation. This has lead to the need for reliable and specific methods of meat species determination in a variety of products, including comminuted, mixed and cooked products. A variety of analytical approaches for meat species identification described in the literature, can be broadly classified into 2 groups: protein-based approaches including electrophoresis and immunoassay and molecular biology-based approaches using DNA.

Traditional

methods

of

meat

authentication,

involving

protein

electrophoresis

or

immunoassay of proteins are largely unsuccessful with heat processed foods because of denaturation and precipitation of proteins. DNA techniques dependent on PCR, have been shown to be successful due to better stability of DNA to heat processing in comparison to proteins. PCR is highly specific and can be exquisitely sensitive for target DNA, but is dependent on appropriate experimental design. DNA is also fragmented by extreme heat and pressure.

Real time quantitative PCR (Q-PCR) is a recent technological advance

allowing the real-time visualisation of amplicon production and as a consequence allowed PCR to be used quantitatively for the first time. Q-PCR may be performed using a number of platforms, the most popular of which is the Taqman system. Taqman technology uses PCR coupled to fluorogenic probes and relies on characterising the reactions by the point in time during cycling when amplification of the PCR product is first detected rather than by the

7

Results and discussion amount of amplicon accumulated after a fixed number of cycles.

The higher the initial

starting copy number of the template DNA, the sooner a significant increase in fluorescence is observed. After comparison with a standard curve it is possible to determine the amount of target DNA in the reaction. This technology is at the forefront of analytical techniques for food authentication. The Food Labelling and Standards Division of the Food Standards Agency (formally of MAFF) funded a number of laboratories to design and optimise a variety of these assays for the 5 most important food species currently available in the UK: turkey, chicken, pork, beef and lamb (Garrett, et al., 2003: Popping and Hird, 2004: Saunders, 2003: and Wiseman, et al., 2003).

The application of the assays and technology transfer to

enforcement and other analytical laboratories, however, has yet to be fully realised. Final evaluation and validation of the assays is therefore needed before technology transfer is appropriate.

This project focused on the final validation of these assays and the

identification of a DNA extraction and purification protocol suitable for the extraction of DNA from a variety of meat matrices. It also sought to develop real-time PCR assays suitable for the quantification of meat in mixtures by investigating calibration methods. The project has provided the Food Standards Agency with a series of protocols in the form of Standard Operating Procedures (SOPs) for the qualitative determination of species present in complex foods. These SOPs are now available in an accessible format to assist enforcement and for use by other analytical laboratories. This outcome serves to move the output from R&D projects from research laboratories into the public domain and for the first time support the use of real-time-PCR based testing methods in the wider analytical community.

RESULTS AND DISCUSSION Validation and comparison of qualitative real-time PCR assays Introduction The aim of the first phase of the project was to identify a DNA extraction protocol appropriate for the extraction and purification of high quality DNA, in the absence of inhibitors, from raw and canned meats. Five DNA extraction techniques, drawn from the collaborating contractors, were tested using raw and canned samples of the five meat species (pork, beef, lamb, chicken and turkey). The DNA extraction protocols included the SDS/proteinase K/Wizard cleanup protocol of RHMT, the Chelex/proteinase K protocol of VLA, the CTAB/proteinase K/Wizard protocol of Eurofins, the CTAB/proteinase K/Sigma

8

Results and discussion GeneElute protocol of CSL and a new method using magnetic beads from Tepnel (Appendix 2). Three of the collaborating contractors (CSL, Eurofins and RHMT) used the protocols on each of the samples. These extracts were then distributed to all contractors so that each received 100 DNA extracts. The extracts were compared for the efficiency of extraction (DNA concentration in mg/ml), the size of DNA fragments determined by agarose gel electrophoresis and the presence of inhibitors determined using either an in-house internal positive control assay or an Applied Biosystems IPC kit (30, 100 and 300 fold dilutions, in duplicate). Additionally, in-house species specific real-time PCR assays were performed on each sample to give a clear indication of extraction efficiency for each of the protocols.

DNA quantification/extraction efficiency The concentration of DNA was measured by each contractor using one of 2 methods: either the UV absorbance of the solution or the incorporation of a fluorescent DNA-intercalating dye. The measurement of the UV absorbance gives the total amount of nucleic acid present whereas the incorporation of fluorescent DNA-intercalating dye measures the double stranded DNA. Comparison of the measurements of DNA for the extracts was highly variable between contractors (figure 1) with a wide range of concentrations quoted for the same extract. For example, the concentration of DNA in the raw chicken sample extracted using the CTAB/proteinase K/Wizard protocol varied between 0.115 and 0.325mg/ml, representing a two fold difference in extracted DNA, for the same sample. The range of concentrations was particularly wide for the chelex extraction protocol, where the concentrations varied between zero and 0.8mg/ml for the same extract. The apparent discrepancy in concentrations between measurements of the same extract by different contractors could be partly accounted for by the different methods of measuring the concentration of DNA.

The

concentrations reflected the method used to measure the DNA present, with generally higher readings derived from the UV absorbance measurements (contractors CSL and CCFRA).

This method measures all nucleic acid present, including fragmented and single

stranded DNA and thus might overestimate the amount of high quality double stranded DNA present; whereas the intercalating dye measurements (RHMT and Eurofins) would not include RNA or single stranded DNA and might therefore, give a better indication of the amount of intact double stranded DNA present in the extracts. This does not hold true however for the chelex extraction protocol, where the DNA in the extracts is recovered as

9

Results and discussion Figure 1 Quantity of DNA extracted from raw and canned meats using 5 different extraction protocols measured by each contractor.

raw

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

CTAB, Proteinase K, Genelute

canned

Chelex SDS, proteinase K, Wizard CTAB, Proteianse K, Wizard

A VL A

C FR

C

C SL

R H M T Eu ro f in s

A VL A

C FR

C

C SL

Tepnel beads


mg/ml

Cow

Measuring contractor

raw

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

canned

CTAB, Proteinase K, Genelute Chelex SDS, proteinase K, Wizard CTAB, Proteianse K, Wizard

VL A

C C FR A

C SL

T

of in s

Eu r

R H M

VL A

C SL

C C FR A

Eu r

R H M

of in s

Tepnel beads

T

mg/ml

Pig


raw

CTAB, Proteinase K, Genelute

canned

Chelex SDS, proteinase K, Wizard CTAB, Proteianse K, Wizard


10

VL A

A C FR

C

C SL

VL A


C

C FR

A

Tepnel beads

C SL

1.2 1 0.8 0.6 0.4 0.2 0


mg/ml

Lamb

Results and discussion Figure 1 continued. Quantity of DNA extracted from raw and canned meats using 5 different extraction protocols measured by each contractor. Chicken raw

mg/ml

1

canned

CTAB, Proteinase K, Genelute Chelex

0.8 0.6

SDS, proteinase K, Wizard CTAB, Proteianse K, Wizard

0.4 0.2

Tepnel beads

A VL A

C FR

C

C SL


A VL A

C FR

C

C SL


0


Turkey raw

1

canned

CTAB, Proteinase K, Genelute Chelex

mg/ml

0.8

SDS, proteinase K, Wizard

0.6

CTAB, Proteianse K, Wizard

0.4

Tepnel beads

0.2

VL A

C SL

C C FR A

Eu r

of in s

T R H M

VL A

C SL C C FR A

of in s

Eu r

R H M

T

0


single stranded confounding measurements using intercalating dyes leading to an under estimate of the amount of potentially double stranded DNA in the sample. Gel electrophoresis on DNA extracted from single meat matrices Each contractor carried out agarose gel electrophoresis on each sample and then assessed the quality and quantity of DNA present. The analysis of the DNA present was then scored for level of fragmentation and quantity of DNA present. These data, however, proved to be too subjective to allow comparison between the samples and therefore this data was not used to determine the most appropriate extraction protocol for DNA from meat matrices. Inhibition assays on DNA extracted from single meat matrices Each contractor assessed the level of inhibitors present in the samples using their in-house assay (Appendix 1). Each duplicate extract was run in triplicate at 3 dilutions: 1 in 30, 1 in 100 and 1 in 300 and the presence and level of inhibitors assessed according to internal

11

Results and discussion standards (Table 1). These assays gave varying results. Some assays detected little or no inhibition in any of the samples whereas others detected inhibitors in the majority of the samples. The assay used by CCFRA detected no inhibitors in any of the samples and those Table 1. Dilution at which PCR inhibitors were detected in DNA extracts of samples of raw and canned meat samples assessed using the in-house assays from the contractors. Extraction protocol CTAB, Proteinase K, Genelute

Matrix treatment Raw

Canned

Chelex

Raw

Canned

SDS, Proteinase K, Wizard

Raw

Canned

CTAB, Proteinase K, Wizard

Raw

Canned

Tepnel beads

Raw

Canned

Species Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig Chicken Turkey Sheep Cow Pig

RHMT -*/30/-/-/-/-/-/-/-/-/-/-/30/30 -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-

Contractor Inhibition assay Eurofins CSL CCFRA -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/30/30 -/-/-/-/-/-/-/30/100/100 -/-/-/-/-/100/100 -/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-/30/30 -/-/30/30 -/-/-/-/-/30/30 -/-/30/30 -/-/30/30 -/-/30/30 -/-/-/-/-/30/30 -/-/-/-/-/-/-/-/-/-/-/30/30 -/-/-/-/-/30/30 -/-/30/30 -/-/-/-/-/-/-/-/30/30 -/-/30/30 -/-

* - denotes no inhibitors detected at the lowest dilution (30 fold). 12

VLA -/-/-/30/-/-/-/-/-/30/300/100/300/300/100/300 -/300/300/100/300/30/30/-/-/-/30/30/-/-/100/300/100/300 100/100/300 100/300 ////100/300 30/30/100/30/-/-/-/-/-/100/-

Results and discussion used by Eurofins and RHMT detected inhibitors at a 1 in 30 dilution in only one and two of the samples, respectively. The assay used at VLA detected inhibitors at the highest dilutions in the greatest number of samples and was considered to be the assay with the greatest sensitivity to PCR inhibitors. In broad terms the results of these assays indicated that PCR inhibitors had been coextracted with the DNA, and that some of the protocols were better at removing these inhibitors than others. On the whole, the CTAB, proteinase K Wizard protocol produced DNA extracts free of inhibitors and the Chelex protocol produced extracts with the greatest concentration of inhibitors, in the majority of the samples. The extracts produced by the CTAB, Proteinase K, Wizard and Tepnel Bead protocols were both found to contain inhibitors using the CSL and VLA assays, however the results of the assays did not always agree and of the 17 samples which were found to contain PCR inhibitors by CSL and/or VLA, in only 6 cases did both assays detect inhibitors in the sample and in the remaining 9, inhibitors were detected by either CSL or VLA alone.

Additionally only the VLA assay

detected inhibitors in the SDS, Proteinase K, Wizard samples. Species-specific real-time PCR assays Each of the extracts was used as a template in a species-specific real-time PCR assay to give an indication of the amount of amplifiable DNA extracted by each of the protocols. Each contractor ran all the samples from a single species (Figures 2 and 3). The results of these assays are clear-cut and indicate that the greatest quantity of amplifiable DNA was consistently extracted using the CTAB, Proteinase K, wizard protocol.

For some matrices,

for example raw chicken and raw pork, comparable CT values were derived from 3 of the extraction protocols and in all cases the CT values for the Tepnel bead protocol were found be either comparable or the lowest after the CTAB, Proteinase K, wizard protocol. Conclusion When all the data was considered it was concluded that the CTAB, Proteinase K, Wizard extraction procedure consistently gave high quality DNA extracts with the greatest quantity of amplifiable DNA. This extraction procedure would be the protocol of choice for extracting DNA from single species meat matrices.

13

C

K, G

en el ut e

SD C S, he pr le ot x ei na se C K, TA W B, iz pr ar ot d ei na se K, W iz ar d Te pn el be ad s

TA B, pr ot ei na se

Ct Value

C

K, G en el ut e



Ct Value

C

K, G en el ut e



Ct Value

Results and discussion

Figure 2 CT values returned by species-specific real-time PCR assays for DNA extracted from raw meat matrices by 5 different extraction protocols. Raw pig 50

40

30

20

10

0

Raw sheep 30

20

10

0

Raw cow 40

30

20

10

0

14

Results and discussion Figure 2 continued CT values returned by species-specific real-time PCR assays for DNA extracted from raw meat matrices by 5 different extraction protocols. Raw chicken

Ct Value

30.00 20.00 10.00


C


K, G

en el ut e

0.00

Raw turkey

Ct Value

50.00 40.00 30.00 20.00 10.00 SD C S, he pr le ot x ei na se C K, TA W B, iz pr ar ot d ei na se K, W iz ar d Te pn el be ad s

C


K, G

en el ut e

0.00

15

C

K, G

en el ut e



Ct Value

C

K, G en el ut e



Ct Value

C

K, G en el ut e



Ct Value


Figure 3 CT values returned by species-specific real-time PCR assays for DNA extracted from canned meat matrices by 5 different extraction protocols. Canned pig 50

40

30

20

10

0

Canned Lamb 30

20

10

0

Canned cow 40

30

20

10

0

16

Results and discussion Figure 3 continued. CT values returned by species-specific real-time PCR assays for DNA extracted from canned meat matrices by 5 different extraction protocols. Canned chicken

Ct Value

40.00 30.00 20.00 10.00


C


K, G

en el ut e

0.00

Canned turkey

Ct Value

50.00 40.00 30.00 20.00 10.00 SD C S, he pr le ot x ei na se C K, TA W B, iz pr ar ot d ei na se K, W iz ar d Te pn el be ad s

C


K, G

en el ut e

0.00

Comparison of extraction protocols for high quality DNA from complex matrices. Introduction The second phase of the project involved the analysis of extraction procedures used to extract DNA from complex mixtures since this was thought to differ from the optimum extraction procedures for pure meat samples. Four mixes of pork and chicken with either rusk or fat were used (Table 2) and additionally a 100% pure pork sample was included to allow comparison of the mixed and pure meat results.

17


Table 2. Composition of complex meat samples

Sample number

Complex meat sample

1.

100% pork

2.

50% pork, 50% chicken and rusk

3.

50% pork, 50% chicken and fat

4.

Equal mix of 2 and 3

5.

100% chicken and rusk

Three of the collaborating contractors (CSL, Eurofins and RHMT) used the protocols from the first phase of the project on each of the samples. These extracts were then distributed to all contractors so that each of the participating contractors received 100 DNA extracts. In the first phase of the project each contractor had performed all the assays, however the data did not always agree and it was difficult to find a consensus for some of the assays. Therefore, to standardise results, the assays were divided amongst the contractors. The concentration of the DNA was determined spectrophotometrically by CCFRA and by fluorescent techniques by Eurofins. Agarose Gel analysis of all samples was carried out by CSL. Two assays, (VLA and RHMT), were used to assess PCR inhibition in all sample extracts and a consensus dilution nominated for each extract after comparison of the results. All contractors then used these dilution factors for chicken or pork specific, or general meat, real-time PCR assays carried out on all extracts. Inhibition assays on DNA extracted from complex meat matrices In order to optimise PCR assay conditions for each sample, the effect of diluting the DNA extracts to reduce the concentration of inhibitors was investigated using the respective inhouse PCR methods. Complex meat mixtures may contain compounds, not completely removed during DNA extraction and clean-up and which may inhibit the PCR. Dilution of DNA samples, and thus dilution of inhibitors, to a level where inhibitory effects are no longer seen allows DNA amplification to occur with maximum efficiency during the PCR.

18

Results and discussion VLA and RHMT used in-house inhibition assays to determine the optimum dilution of DNA to use in PCR analysis of each sample. Very similar results were obtained using the two inhibition assays. DNA extracted from raw meat samples using the Chelex protocol showed the highest levels of inhibition, therefore these DNA extracts were diluted the most prior to PCR analysis (1/300 or 1/1000 depending on the complex meat sample). For all other extracts, identical DNA dilutions (1/30) were recommended as showing no inhibition. Gel electrophoresis on DNA extracted from complex meat matrices The samples were subjected to electrophoretic fractionation using agarose gels and the quality and quantity of DNA present, assessed (Plate 1). All the DNA extractions gave electrophoretic profiles except the chelex extractions, however this was expected since this extracts contain single stranded DNA which cannot be visualised on agarose gels stained with the interchelating dye ethidium bromide. Quantification of DNA extracted from complex meat matrices Replicate DNA extracts were quantified and an estimate of the DNA purity determined using the 260:280 ratio. Quantification was performed using a GeneQuant pro DNA calculator. Full details of the method used are given in the appendix. The results of quantifying DNA extracts from raw and canned meat complexes using spectrophotometric measurements are shown in Table 3a and 3b respectively. Without applying a full statistical analysis to the results of DNA extraction from raw complexes, it appears that there is little significant difference between the concentration (µg/ml) of DNA extracted from the different products with each of the extraction methods. Also, although the Chelex method appears to produce the highest yield of DNA, the purity of this is low (260:280 35). If the negative control failed (CT