Methodologies Basmati Rice & Bushmeat

Methodologies
Basmati Rice &
Bushmeat
DNA Seminar,
Thursday 8 th May 2008

Basmati rice varieties
• Saving on EU import duty of €65 per tonne for defined premium
basmati varieties from India and Pakistan.
• Premium price
• Rice Association Code of Practice (July 2005) max limit for nonbasmati
rice is 7% in products labelled as “basmati”.
• In 2003 the main adulterants were Sherbati and Pak 386, detected in
46% of products.
• In 2006, adulteration with these two varieties had decreased to 16%

Intensity
Existing authentication method
• Based on the use of ‘microsatellite’ markers (= SSRs, STRs !)
• Size of markers used to create a variety-specific profile
• Area under the curve used to quantify levels of adulterant varieties
Size

Agilent project
• Aim to convert existing tests onto a single platform
• Agilent 2100 BioAnalyzer
• Difficulties in using microsatellite markers
• Resolution >15 base pairs
• Difficulties in measuring area under curve
• Solution: alternative marker type required

Indel markers
3’ 5’
3’ 5’
Size range of insertion/deletion is between 100 bp
Generate large differences in fragment size
Readily converted to PCR markers with 2 co-dominant alleles
400,000 available in the rice genome database:
• One INDEL every 1k bp
• Flexible choice of fragment size based on primer design

Assay design
• Examine the size of fragments
across multiple varieties and
multiple indels
Taraori
• Isolate indels that show
categorical differences among
varieties
Sherbati
• Select a panel of markers to
produce a variety-specific
profile
Size

Size
Example output
1 2 3 4 5 6 7 8 9 10 11 12
P 1 N N N P N 2 N N N P N P
9
8
7
6
5
4
3
2
1

Variety results – ID rules
• 596 (550-600) control (present in all)
• 354 (330-358) all non-fragrant varieties
• 273 (270-274) present in all approved Basmatis
• 256 (254-260) *
• 214 (213-218) present in all approved Basmatis
• 186 (181-187) *
• 174 (171-175) *
• 142 (141-144) all approved except Pusa & Haryana
• * if all 3 are present the sample contains a non-permitted
variety

Band profiles
Basmati Group 1 B1 B5 B4 B7
Pusa Basmati (Group 2)
Confirm with follow-on test I5
Basmati 385 (Group 3)
Confirm with follow-on test I10
B2 B3 B4 B7
B1 B3 B4 B7
Kasturi (Group 4) B1 B5 B6 B7
Haryana Basmati (Group 5) B2 B5 B6 B7
Mahi Sugandha (Group 6) B1 B3 B6 B7
Adulterant varieties
Pak 386 B1 B3 B4 B8
Sherbati 1 B2 & B1 B3 B4 B8
Sherbati 2 B2 B3 B6 B8
Supra B1 B3 B6 B7 & B8
Superfine B2 B3 B6 B7 & B8
Mugad Sugandha B2 B3 B6 B7 & B8
Pusa Sugandha B2 B3 B6 B7

Variety SOP
FOOD STANDARDS AGENCY
STANDARD OPERATING PROCEDURE (SOP) 3
Version 1.0, October, 2007
STANDARD OPERATING PROCEDURE FOR THE DETECTION OF
NON-PERMITTED RICE VARIETIES BY INDEL-PCR ANALYSIS
USING THE AGILENT 2100 BIONALYZER
• Available for use in-house
• Available as a service from Tepnel and other genetic analysis providers

Quantitative test
• Original test uses area under curve
• Preferred DNA quantification of mixtures
is via Real-Time PCR
• Agilent method restricted to analysis of
electrophoresis peaks
• Decision to be made regarding baseline /
threshold for measurement
• Has implications for quantitative analysis

Quantitative SOP
FOOD STANDARDS AGENCY
STANDARD OPERATING PROCEDURE (SOP) 1
Version 1.0, August, 2007
STANDARD OPERATING PROCEDURE FOR THE QUANTITATIVE
ANALYSIS OF ADULTERATION OF BASMATI RICE WITH THE
VARIETIES SHERBATI, MUGAD SUGANDHA, PAK 386 OR SUPERFINE
USING THE AGILENT 2100 BIONALYZER AND A MICROSATELLITE
PCR MARKER
• Recommended as an initial screen for adulteration above 7%
• Quantification with these methods is complicated, assumptions
need to be understood
• For legal enforcement, the original test should be used.

Bushmeat Identification
• Surveys at Heathrow and London markets reveal bushmeat
importation
• Issues: Zoonoses, livestock disease, meat hygiene, conservation
Southwark market.
Smoked antelope meat.
Smoked cane rat.

Species identification
• Species identification of truly unknown samples
requires DNA sequencing
• Sequencing is not possible at most PA labs
• Species detection tests easier to perform
• Test exist for many common species exist
• Aim to develop a test covering a range of illegally
imported African wild species:
• Chimpanzee, Gorilla, Mandrill
• Duiker, Bushbuck, Porcupine
• Cane rat, Grass cutter, Pouched rat
• Bush pig, Zebu, African sheep

Methodology
Restriction Fragment Length Polymorphism (RFLP)
• Amplify common fragment by PCR (mitochondrial cytochrome b gene)
• Digest with two restriction enzymes
• Generate fragment profile to match against references
HinfI
MseI
Species A
Species B
Species C

Chimpanzee
Chimpanzee
Gorilla
Gorilla
Bushbuck
Bushbuck
African sheep
African sheep
Dwarf zebu
Dwarf zebu
Zebu
Zebu
Example results
• Agilent output: fragments (bands) following HinfI restriction
• Different species show
different numbers and
size of fragment
• Where a pattern is
shared between species,
different enzymes may
be used

Example results
• Fragment categories define profiles
Species
HinfI
MseI
Chimp C E K D E L
Gorilla G M H L
Bush buck E G K I L
Sheep E N E F K
Dwarf zebu B E K J K
Zebu B E L J K
Duiker 1 C E K D G K
Duiker 2 C E K E M
Bush Pig D E K C E L
Pouched Rat E N D E L
Fragment size Category Fragment size Category
1 to 20 A 155 to 180 H
25 to 40 B 185 to 200 I
45 to 60 C 205 to 225 J
65 to 90 D 230 to 260 K
95 to 105 E 325 to 345 L
110 to 125 F 350 to 370 M
Cane 130 to Rat 150 E M G 375 to 400D F G NH K
Porcupine C E F K D E G H

Applications
• Best to use following negative results from common species
identification tests
• Care needs to be taken to ensure complete digestion
• Care needs to be taken when ‘binning’ bands (size variation)
• SOP available from FSA:
FOOD STANDARDS AGENCY
STANDARD OPERATING PROCEDURE (SOP) 1
Version 1.3, November, 2007
STANDARD OPERATING PROCEDURE FOR THE GENETIC
IDENTIFICATION OF COMMON AFRICAN WILDMEAT SPECIES
USING THE AGILENT 2100 BIOANALYZER

Acknowledgements
Katherine Steele, Ross McEwing, John Gorham
(Food DNA Services)
Jacqui Coutts
(Tepnel)
rogden@tepnelscientific.com