The use of the Jellett rapid testing kit for PSP toxins detection in the UK statutory monitoring programme for marine biotoxins - Biotoxin Team Report
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Final report The use of the Jellett rapid testing kit for PSP toxins detection in the UK statutory monitoring programme for marine biotoxins Biotoxin Team Report
The use of the Jellett rapid testing kit for PSP toxins
detection in the UK statutory monitoring programme
for marine biotoxins
Final report
26th January 2007
31 pagesCONTENTS
Page
Executive summary ...............................................................................................................3
List of Abbreviations ..............................................................................................................5
PART ONE: Internal QC evaluation of the PSP Jellett Rapid Testing kit batch 40000-
19Apr05
Introduction .....................................................................................................................7
Materials & methods .......................................................................................................8
Results ............................................................................................................................11
Discussion and conclusion..............................................................................................17
Consequential actions.....................................................................................................19
PART TWO: Analysis of field samples for paralytic shellfish poisoning toxins by the
Jellett Rapid Test
Introduction and background ..........................................................................................21
Materials & methods .......................................................................................................22
Results ............................................................................................................................25
Discussion and conclusion..............................................................................................28
References ............................................................................................................................30
Page 2 of 31EXECUTIVE SUMMARY
This report summarises the results and experiences gained by Cefas in 2006
during a period of testing of the Jellett Rapid Test (JRT), a commercial
antibody-based qualitative test developed and manufactured by Jellett Rapid
Testing Ltd as rapid test to be used as a screen for the detection of Paralytic
Shellfish Poisoning (PSP) toxins in bivalve shellfish in the UK statutory
monitoring programme for biotoxins. The test is claimed to detect PSP toxins
above 40 μg/100g shellfish tissue in 35 minutes (manufacturer’s website).
Part One of this report describes the results of an internal quality control (QC)
evaluation conducted on the PSP JRT batch 40000-19Apr05. The batch was
received by Cefas in February 06 having been recertified by Jellett Rapid
Testing Ltd until January 2007 (i.e. shelf life extended by 8 months). The
certificate of analysis (CoA) of the batch indicated that it had passed the
manufacturer’s own quality control checks. The batch was submitted to
additional internal quality controls (as described in this report) to establish its
response to all PSP toxins since the information provided by the CoA related
to only two of the PSP toxins (namely neosaxitoxin (NEO) and saxitoxin
(STX)) and did not provide any information on the limits of detection (LOD) of
the batch for specific PSP toxins. A total of five quality control checks were
conducted at the laboratory to assess the response of the batch to purified
toxins (namely STX, NEO, gonyautoxins (GTX) 1,4 together, 2,3 together, 5
(also known as B1) and decarbamoyl saxitoxin (dcSTX)). The response of the
batch to mixtures of toxins was also assessed, since it had been suggested
that the JRT kit performs better in the presence of toxin mixtures.
Results showed that batch 40000-19Apr05 was able to detect STX, dcSTX,
GTX2/3 and GTX5 alone at concentrations well below the regulatory limit of
80 μg STX eq./100g. However the batch was found to have a very low
sensitivity to NEO and GTX1/4. The kit’s response to GTX1/4 in particular
was very significantly in excess of the regulatory limit. The reactivity
established to NEO and GTX1/4 could not be reconciled with the
manufacturer’s claim that the kit can detect “all saxitoxins as low as
40µg/100grams”. QC checks conducted on shellfish material spiked with
mixtures of PSP toxins showed that batch 40000-19Apr05 also failed to detect
several samples containing toxin mixtures at levels above the stated kit and
batch LOD of 40 μg/100g. In addition to the limitations of the batch with regard
to GTX1/4 alone tests containing approximately 40 μg/100g of PSP toxins
made up of GTX1/4 in combination with STX were also not detected, both
when GTX1/4 was predominant but also at the ratio of 50:50. Two samples
were not detected by 40000-19Apr05 at the regulatory limit. One test
contained 95% GTX1/4 and 5% STX and the second, representative of a toxin
profile seen in a mussel sample during a large UK PSP incident in 1990,
consisted of NEO in isolation.
Batch 40000-19Apr05 was not considered as abnormal by Jellett Rapid
Testing Ltd but Cefas results led to the UK National Reference Laboratory
(NRL) recommending suspension of the use of the batch in the UK official
Control biotoxin monitoring programme and to the issue of a UKNRL standard
Page 3 of 31operating procedure (SOP) detailing the QC performance criteria checks to be
conducted prior to the use of the JRT for PSP by Official Control laboratories.
Batch 40000-19Apr05 and subsequent JRT kit batches (16Feb06 and
21Jul06) received by Cefas have not complied with the requirement laid out in
the UKNRL SOP for JRT QC and have therefore not been used for official
control testing for PSP in the UK.
Part Two of this report describes the experiences gained with the JRT and the
results obtained with the kit between 26th May and 31st October 2006, when
the kit was incorporated in a trial aiming to assess the suitability of High
Performance Liquid Chromatography (HPLC) as an alternative to the
European reference method for PSP testing, the biological assay (MBA). Four
batches of JRT kits were used during the course of the trial; none of them
complied with the quality assurance criteria specified in the UKNRL SOP for
use in OC testing. JRT test results reported here were therefore not used for
OC purposes.
A total of 961 shellfish samples consisting of Common mussels (Mytilus
edulis) (640), Pacific oysters (Crassostrea gigas) (142), Native oysters
(Ostrea edulis) (101), Queen scallops (Aequipecten opercularis) (17), Razor
clams (Ensis sp.) (6), Common cockles (Cerastoderma edule) (51), Hard
clams (Mercenaria mercenaria) (3) and King scallops (Pecten maximus) (1),
originating from the Scottish, English, Welsh, Isle of Man and Isle of Jersey
biotoxin statutory monitoring programmes were included in the trial.
Results were as follows:
• The subjective nature of the assay scoring was found to complicate test
interpretation and could lead to variable interpretation particularly around
the 50% end-point of the assay. The variability observed in this study
was found to compare well with that described by Mackintosh et al., 2002
[15].
• A proportion of the strips used between May and October 2006 yielded
invalid results. Overall,ABBREVIATIONS USED IN THE TEXT
AL Regulatory Limit (0.8 µg STX equivalent/g shellfish tissue)
AOAC Association of Official Analytical Chemists
B1(GTX5) Gonyautoxin 5
C-1 N-sulfocarbamoyl toxin C1
C-2 N-sulfocarbamoyl toxin C2
C-3 N-sulfocarbamoyl toxin C3
C-4 N-sulfocarbamoyl toxin C4
Cefas The Centre for Environment, Fisheries and Aquaculture
Sciences, UK
CoA Certificate of Analysis
dcSTX decarbomoyl saxitoxin
EU European Union
FSA Food Standards Agency, UK
GTX Gonyautoxin
GTX2/3 Gonyautoxins 2 and 3 together
GTX1/4 Gonyautoxins 1 and 4 together
HPLC High Performance Liquid Chromatography
JRT Jellet Rapid Test for PSP
LOD (lod) Limit of Detection
MBA Biological bioassay
MS Member States
NEO Neosaxitoxin
NG (-ve) Negative
PS (+ve) Positive
PSP Paralytic Shellfish Poisoning
QC Quality Control
SOP(s) Standard Operating Procedure(s)
STX Saxitoxin
UKNRL UK National Reference Laboratory for Biotoxins
Page 5 of 31PART ONE
Internal QC evaluation of the PSP Jellett Rapid Testing
kit batch 40000-19Apr05
Page 6 of 311. Introduction and background
Paralytic shellfish poisoning (PSP) toxins are potent neurotoxins produced by
certain marine dinoflagellates and can accumulate in filter feeding shellfish to
highly toxic levels. Shellfish destined for human consumption are required
under EU (EC Regulation 854/2004 [1]) and national legislation to be routinely
monitored to protect consumer’s health. The reference method specified by
European legislation for Official Control (OC) testing for PSP to protect public
health is the biological assay (MBA).
The Jellett Rapid Test (JRT) is a commercial antibody-based qualitative test
manufactured by Jellett Rapid Testing Ltd, Canada. The test uses polyclonal
PSP toxin antibodies and works on the principle of lateral flow immuno-
chromatography using a strip format (similar to pregnancy kits). The test is
claimed to detect PSP toxins above 40 μg/100g shellfish tissue in 35 minutes
(manufacturer’s website). The JRT test has been developed as a
commercially available rapid test to be used as a screen for PSP detection in
bivalve shellfish.
Analysis involves dilution of a shellfish extract in JRT running buffer, loading
the sample on to a JRT detection strip and waiting for 35 minutes for a colour
reaction to develop. At that time, following the manufacturer’s instructions, the
colour intensity of a test line (or T line) is read against that of a control line (or
C line) to define whether the sample is positive or negative. The schematic
below illustrates positive and negative tests.
C-line
T-line
Positive sample
Negative sample
The JRT kit (formerly, MIST Alert™) was used from January 2005 until May
2006, in conjunction with the MBA, as a qualitative screening test for the
detection of PSP toxins in mussels and King scallops under the scope of the
Scottish OC marine biotoxin monitoring programme.
The use of the PSP JRT kit by UK monitoring laboratories is controlled by a
UK National Reference Laboratory (UKNRL) Standard Operating Procedure
(SOP) for the analysis of shellfish extracts by JRT. Version 1 of this SOP
Page 7 of 31included provisions for the performance of internal quality control (QC) checks
to be performed on each new box and each new batch of kit using archived
negative and positive (naturally contaminated) shellfish extracts (as
determined by MBA). The quality of each batch is also assessed by Jellett
Rapid Testing Ltd and certificates of analysis (CoA) are issued with each
batch. The certificate states that “several concentrations of STX and NEO
were tested on the batch” and that “the lower limits of detection and the
concentrations at which the T-line is visibly present or absent are consistent
with previously released lot of the same type”. The batch limits of detection
(LOD) for STX or NEO are not specified on the CoA. The response of the
batch to other PSP toxins is not described.
A stock of PSP kit from batch 40000-19Apr05 was purchased by Cefas for the
purpose of the Scottish marine toxin monitoring programme in February 2006
after its shelf life was extended by the manufacturer from April 06 to January
07. After discussions with the Food Standards Agency (FSA), the UK
Competent Authority responsible for the implementation of EU and national
legislation on food safety including that of the national marine biotoxin
statutory monitoring programme, it was agreed that additional independent
QC should be conducted by Cefas on this batch to confirm the stability of its
response towards PSP toxins.
This report summarises the results of the QC checks (6 separate checks in
total) performed by Cefas on JRT batch 40000-19Apr05 using shellfish
extracts spiked with either individual PSP toxins or mixtures of PSP toxins at
various concentrations.
2. Materials and methods
Toxin standards
Certified reference standard solutions of saxitoxin (STX), neosaxitoxin (NEO),
gonyautoxins 2 and 3 together (GTX2/3), gonyautoxins 1 and 4 together
(GTX1/4), decarbomoyl saxitoxin (dcSTX) and gonyautoxin 5 (GTX5, also
known as B1) were purchased from the Institute of Marine Biosciences
National Research Council of Canada (NRC-IMB).
Shellfish extract
Mussel extracts, determined to be negative for PSP by the MBA and negative
by JRT were selected for use in all QC checks reported here. One mussel
extract was used for all QC checks carried out with individual toxins. A second
extract was used for the work on toxin mixtures. Both extracts were from
Common mussel (Mytilus edulis) samples submitted to the laboratory for PSP
toxin testing as part of the national biotoxin monitoring programme. Tissue
extraction and MBA testing had been carried out according to established
Cefas SOPs and HCl extracts kept frozen until needed. Cefas procedures for
extraction and testing for PSP toxins by MBA are based on the AOAC method
959.08 [2] and the test method is accredited by UKAS under ISO 17025.
Page 8 of 31Jellett Rapid Test
Strips were selected at random from JRT batch 40000-19Apr05 purchased
from Jellett Rapid Testing Ltd in February 06. Buffer reference 40004-
04Jan06 was supplied by the manufacturer with the above batch of strips.
Both PSP test strips and saline buffers were provided with CoA showing that
they had passed the manufacturer’s QC. The response of the kit to “several
concentrations of STX and NEO” was stated to be “consistent with the
previously released lot of the same type”.
Preparation of standards
Each certified toxin standard solution of known concentration was diluted to a
set working concentration using 0.1M acetic acid. Working concentrations for
each toxin standard were adjusted according to relative toxicity compared with
STX. The molecular weights and relative toxicities (NRC PSP Supplemental
Information [3] after Oshima, 1995 [4]) used in calculations of toxin
concentrations and actual concentrations at the regulatory limit (AL) are given
in Table 1. For simplicity in this report toxin concentrations are expressed in
relation to the AL rather than according to the actual concentration of toxin
present. For GTX1/4 and GTX2/3, the concentrations are summed, but the
relative toxicities considered for the more toxic component only (i.e.: GTX1
and GTX3, respectively).
Toxin standards were prepared in plastic vials, vortex mixed for 10 sec and
used within 3 hours.
Table 1: PSP standard concentrations at the regulatory limit (AL) and relative toxicities to STX [3,4]
Molecular weight
PSP Toxin (g/Mole) Relative toxicity Conc. at AL
(HCl salt) to STX (µg/g)
NEO 388.2 0.9243 0.87
GTX1 447.8 0.994 0.80
GTX4 447.8 0.7261 1.10
STX 372.2 1 0.80
dcSTX 329.2 0.5131 1.56
GTX5 415.8 0.0644 12.42
GTX2 431.8 0.3592 2.23
GTX3 431.8 0.6379 1.25
Fortification of shellfish tissues with purified toxins
Working concentrations of toxin were added to negative mussel extract to
prepare a series of fortified samples. Dilutions were subsequently made with
blank mussel extract to prepare shellfish extracts spiked with purified toxins at
the range of toxin concentrations given in Table 2. Spiked extracts were
thoroughly mixed by vortex mixing and used within 3-4 hours. Spiked
materials were vortexed prior to use.
Page 9 of 31Table 2: Fortification of shellfish tissues with PSP toxins: toxin concentrations tested in each JRT QC
Qc reference Toxin tested Concentrations tested in spiked mussel extract
Initial concentration Subsequent dilutions
QC 1 STX 1 AL 1/2, 1/4, 1/5 and 1/6 AL
dcSTX 1 AL 1/2 1/5, 1/10 and 1/15 AL
GTX2/3 1 AL 1/2, 1/4, 1/8 and 1/10 AL
GTX5 1/10 AL 1/50, 1/80 and 1/100 AL
GTX1/4 2 AL 1, 1/2, 1/3 and 1/4 AL
QC 2 GTX1/4 12 AL 8, 4, 2 and AL
QC 3 NEO 2 AL 1,1/2,1/3,1/4 and 1/5 AL
QC 4 GTX1/4 12 AL 8 AL
QC 5 NEO 2 AL 1 and 1/2 AL
Fortification of shellfish tissues with toxin mixtures
Mussel extracts were spiked with mixtures of PSP toxins, in the proportion
indicated in Table 3. Fortification of mussel extracts was conducted as
described above. A volume of 0.5 ml of spiked extract was prepared for each
toxin profile at a total toxin concentration of 5 μM. Serial dilutions of these
were then prepared using blank mussel extract to achieve the required test
concentrations listed in Tables 10. Fortified shellfish tissues were assayed
within 3-4 hours and vortexed prior to use.
Table 3: Fortification of shellfish tissues with toxin mixtures: toxin profiles tested
Percentage of toxin in sample
Mixture GTX1/4 STX NEO GTX2/3
1 95 5 0 0
2 90 10 0 0
3 75 25 0 0
4 50 50 0 0
5 25 75 0 0
6 15 7 23 55
7 0 0 100 0
8 9 0 0 91
9 6 0 49 45
10 4 0 48 48
11 21 0 57 22
12 15 0 69 16
13 0 100 0 0
14 100 0 0 0
15 0 0 0 100
Mixtures 1-5 represented ratios previously used and reported by Jellett Rapid
Testing Ltd (confidential information provided by the manufacturer to the FSA
on the response of the Jellett Rapid Test batch 40000-19Apr05 to toxin
mixtures). Mixtures 6-12 reflected toxin profiles detected during the PSP
outbreak which affected the North East coast of England in 1990 (6-7: mussel
samples; 8-12: scallop samples), as determined by HPLC (Cefas, unpublished
data). Samples 13-15 were single toxin spiked materials included in the QC
check as controls.
Page 10 of 31The predicted toxicity of each spiked extract (in μg STX-diHCl equivalents /
100g shellfish flesh) was calculated from the concentrations present (μM), the
molecular weight of the toxins (HCl salt) and their relative toxicities using data
published by NRC (PSP supplemental information [3], after Oshima, 1995 [4])
(Table 1).
It was not possible to spike extracts separately with GTX1/4 or 2/3 using the
reference solutions as they are only supplied as mixtures at a stable ratio. For
mixtures 1-5, GTX 1/4 and 2/3 ratios were those found in the reference
solution. For 6-12, the total concentration (μM) reported in the outbreak
samples was used (i.e. GTX 1&4 were summed, and GTX2&3 were summed).
For the calculation of predicted toxicity of a spiked extract, the relative toxicity
of the GTX2/3 was used, calculated from the relative toxicity of GTX2 and
GTX3 and the levels at which they are present at the stable ratio in the
reference solutions. For GTX2/3 the molar ratio in reference solutions is
118:39, for GTX1/4 the molar ratio is 106:35.
Analytical procedure
Samples were tested in accordance with the UKNRL SOP for the analysis of
shellfish extracts by the Jellett Rapid Test and in compliance with the
manufacturer’s instructions. 400 μl of JRT running buffer was placed into
individual microcentrifuge tubes using a calibrated pipette and 100 μl of test
sample added and mixed by pipetting. 100 μl of the mixture was then placed
onto the sample pad of the JRT detection strip. JRT strips were incubated at
room temperature for 35 minutes prior to reading. Where possible, up to three
replicate strips were used for each test concentration. Experiments on toxin
mixtures could only be performed on one test strip per test concentration due
to a shortage of stocks for batch 40000-19Apr05.
Test interpretation
Results were interpreted according to the JRT instruction sheet supplied with
the batch of kit in February 2006.
Each strip was read independently by four analysts (A, B, C & D) experienced
in JRT interpretation and results recorded. Strips for the toxin mixture work
were read by one senior analyst. A result was considered positive when the
intensity of the T line was 0%, 25% or 50% of that of the C line, and negative
when the intensity of the T line was 75% or 100% of that of the C line, in
accordance with the manufacturer’s instructions and the UKNRL SOP. The
sensitivity end-point (LOD) for each toxin titration was estimated as the point
at which, overall, less than 75% of the inoculated test strips were scored as
positive by the analysts.
3. Results
3.1. Response to single toxins
3.1.1. Saxitoxin (STX)
JRT strips were inoculated with mussel extracts spiked with STX at
concentrations ranging from 1/6 to 1AL. Results are summarised in Table 4.
Page 11 of 31Table 4: Number of positive results scored by four independent analysts at various concentrations of
saxitoxin
Concentration Number of Number of positive results scored by each
STX replicates analyst
A B C D
1AL 1 1 1 1 1
1
/2AL 1 1 1 1 1
1
/4AL 3 3 3 3 3
1
/5AL 3 2 3 3 2
1
/6AL 3 0 1 1 1
STX concentrations of one fifth of the regulatory limit (1/5AL) and above gave
>80% (10 of 12) positive results. At 1/6AL, only 25% (3 of 12) strips were
scored as positive. Therefore the limit of detection (LOD) for STX was
estimated to be between 1/6AL (i.e. 13 μg/100g) and 1/5AL (i.e. 16 μg/100g).
3.1.2. Decarbamoylsaxitoxin (dcSTX)
JRT strips were inoculated with mussel extracts spiked with dcSTX at
concentrations ranging from 1/15 to 1AL. Results are summarised in Table 5.
Table 5: Number of positive results scored by four independent analysts at various concentrations of
decarbamoylsaxitoxin
Concentration Number of Number of positive results scored by each
dcSTX replicates analyst
A B C D
1AL 1 1 1 1 1
1
/2AL 3 3 3 3 3
1
/5AL 3 0 3 3 1
1
/10AL 3 0 0 0 0
1
/15AL 1 0 0 0 0
dcSTX concentrations of half the regulatory limit (1/2AL) and above gave
consistently positive results. A dcSTX concentration of 1/5AL gave variable
results. Different analysts ranged from scoring all three replicates as positive
to scoring all three as negative. Overall 58% (7 of 12) strips were scored as
positive at this concentration. The LOD for dcSTX was therefore estimated to
be between 1/5AL (i.e. 16 μg STX eq./100g) and 1/2AL (i.e. 40 μg STX
eq./100g).
3.1.3. Gonyautoxins 2 and 3 (GTX2/3)
JRT strips were inoculated with mussel extracts spiked with GTX2/3 at
concentrations ranging from 1/10 to 1AL. Results are summarised in Table 6.
Table 6: Number of positive results scored by four independent analysts at various concentrations of
gonyautoxins 2 and 3
Concentration Number of Number of positive results scored by
GTX2/3 replicates each analyst
A B C D
1AL 1 1 1 1 1
1
/2AL 1 1 1 1 1
1
/4AL 3 3 3 3 3
1
/8AL 3 0 2 2 0
1
/10AL 3 0 0 1 0
Page 12 of 31GTX2/3 concentrations of a quarter of the regulatory limit (1/4AL) and above
gave consistently positive results. At a GTX2/3 concentration of 1/8AL, 33% (4
of 12) strips were recorded as positive. The LOD for GTX2/3 was therefore
estimated to be between 1/8AL (i.e. 10 μg STX eq./100g) and 1/4AL (i.e. 20
μg STX eq./100g).
3.1.4. Gonyautoxin 5 (GTX5 or B1)
JRT strips were inoculated with mussel extracts spiked with GTX5 at
concentrations ranging from 1/100 to 1/10AL. Results are summarised in
Table 7.
Table 7: Number of positive results scored by four independent analysts at various concentrations of
gonyautoxin 5
Concentration Number of Number of positive results scored by each
GTX5 Replicates analyst
A B C D
1
/10AL 3 2 3 3 3
1
/50AL 3 1 3 2 1
1
/80AL 1 0 0 0 0
1
/100AL 1 0 0 0 0
GTX5 concentrations of a tenth of the regulatory limit (1/10AL) gave
consistently positive results (>90%). A GTX5 concentration of 1/50AL gave
variable results of 58% positive (7 of 12). The LOD for GTX5 is therefore
estimated to be between 1/50AL (i.e. 1.6 μg STX eq./100g) and 1/10AL (i.e. 8
μg STX eq./100g).
3.1.5. Gonyautoxins 1 and 4 (GTX1/4)
JRT strips were inoculated with mussel extracts spiked with GTX1/4 at
concentrations ranging from 1/4 to 12AL. A number of strips were also
subsequently inoculated with 100 μl of pure GTX1/4 standard from the
ampoule provided by NRC mixed with JRT buffer. Results are summarised in
Table 8.
Table 8: Number of positive results scored by four independent analysts at various concentrations of
gonyautoxins 1 and 4
QC Concentration Number of Number of positive results scored by each
Reference GTX1/4 Replicates analyst
A B C D
2 15.2AL (pure 3 0 2 2 2
4 standard from 3 0 1 1 0
ampoule)
2 3 0 0 0 0
12AL
4 3 0 0 0 0
2 1 0 0 0 0
8AL
4 3 0 0 0 0
2 4AL 1 0 0 0 0
2 1 0 0 0 0
2AL
1 3 0 0 0 0
2 1 0 0 0 0
1AL
1 3 0 0 0 0
1
1 /2AL 1 0 0 0 0
1
1 /3AL 1 0 0 0 0
1
1 /4AL 1 0 0 0 0
Page 13 of 31None of the spiked mussel samples yielded any response with the test,
indicating that the LOD for GTX1/4 was greater than 12AL. When inoculated
with a toxin standard removed directly from the NRC ampoule, a positive
response was noted by some analysts on 8 of 24 occasions (33%). This
suggests that the LOD of the JRT test for GTX1/4 is greater than 15.2AL (i.e.
>1,216 μg STX eq./100g).
3.1.6. Neosaxitoxin (NEO)
JRT strips were inoculated with mussel extracts spiked with NEO at
concentrations ranging from 1/5 to 2AL. Results are summarised in Table 9.
Table 9: Number of positive results scored by four independent analysts at various concentrations of
Neosaxitoxin
QC Concentration Number of Number of positive results scored by
Reference NEO replicates each analyst
A B C D
3 3 3 3 2 1
2AL
5 3 0 2 0 0
3 3 1 1 1 1
1AL
5 3 0 0 0 0
3 1 3 0 0 0 0
/2AL
5 3 0 0 0 0
1
3 /3AL 1 0 0 0 0
1
3 /4AL 1 0 0 0 0
1
3 /5AL 1 0 0 0 0
NEO concentrations at 2AL were not consistently positive with only 46% of
strips returning positive results (11 of 24) over two testing occasions. Results
varied with 75% (9 of 12) positive on QC run 3 and 6% (2 of 12) positive on
QC run 5. At toxin concentrations of 1AL and below strips were consistently
negative (8 0.59 0.03 0.00 0.00 43.3 NG
16 0.30 0.02 0.00 0.00 21.7 NG
2 2 2.25 0.25 0.00 0.00 174.0 PS
4 1.13 0.13 0.00 0.00 87.0 PS
8 0.56 0.06 0.00 0.00 43.5 NG
16 0.28 0.03 0.00 0.00 21.7 NG
3 2 1.88 0.63 0.00 0.00 176.0 PS
4 0.94 0.31 0.00 0.00 88.0 PS
8 0.47 0.16 0.00 0.00 44.0 NG
16 0.23 0.08 0.00 0.00 22.0 NG
4 2 1.25 1.25 0.00 0.00 179.4 PS
4 0.63 0.63 0.00 0.00 89.7 PS
8 0.31 0.31 0.00 0.00 44.8 NG
16 0.16 0.16 0.00 0.00 22.4 NG
5 2 0.63 1.88 0.00 0.00 182.7 PS
4 0.31 0.94 0.00 0.00 91.4 PS
8 0.16 0.47 0.00 0.00 45.7 PS
16 0.08 0.23 0.00 0.00 22.8 NG
Table 10b: Results of JRT QC checks performed using mussel extracts spiked with assorted toxin levels and
profiles – Profiles described during the UK 1990 PSP outbreak
Predicted
Toxin concentration (in µM)
Dilution toxicity (in JRT Result
Mixture
factor µg STX PS-NG
GTX1/4 STX NEO GTX2/3
eq./100 g)
6 2 0.38 0.18 0.58 1.38 122.3 PS
4 0.19 0.09 0.29 0.69 61.2 PS
8 0.09 0.04 0.14 0.34 30.6 NG
16 0.05 0.02 0.07 0.17 15.3 NG
7 2 0.00 0.00 2.50 0.00 172.0 PS
4 0.00 0.00 1.25 0.00 86.0 NG
8 0.00 0.00 0.63 0.00 43.0 NG
16 0.00 0.00 0.31 0.00 21.5 NG
8 2 0.23 0.00 0.00 2.28 88.1 PS
4 0.11 0.00 0.00 1.14 44.0 PS
8 0.06 0.00 0.00 0.57 22.0 PS
16 0.03 0.00 0.00 0.28 11.0 PS
9 2 0.15 0.00 1.23 1.13 130.5 PS
4 0.08 0.00 0.61 0.56 65.3 PS
8 0.04 0.00 0.31 0.28 32.6 NG
16 0.02 0.00 0.15 0.14 16.3 NG
10 2 0.10 0.00 1.20 1.20 127.7 PS
4 0.05 0.00 0.60 0.60 63.9 PS
8 0.03 0.00 0.30 0.30 31.9 NG
16 0.01 0.00 0.15 0.15 16.0 NG
11 2 0.53 0.00 1.43 0.55 151.8 PS
4 0.26 0.00 0.71 0.28 75.9 PS
8 0.13 0.00 0.36 0.14 38.0 PS
16 0.07 0.00 0.18 0.07 19.0 NG
12 2 0.38 0.00 1.73 0.40 157.3 PS
4 0.19 0.00 0.86 0.20 78.7 PS
Page 15 of 318 0.09 0.00 0.43 0.10 39.3 NG
16 0.05 0.00 0.22 0.05 19.7 NG
Table 10c: Results of JRT QC checks performed using mussel extracts spiked with assorted toxin levels and
profiles – toxin controls
Predicted
Toxin concentration (in µM)
Dilution toxicity (in JRT Result
Mixture
factor µg STX PS-NG
GTX1/4 STX NEO GTX2/3
eq./100 g)
13 2 0.00 2.50 0.00 0.00 186.1 PS
4 0.00 1.25 0.00 0.00 93.1 PS
8 0.00 0.63 0.00 0.00 46.5 PS
16 0.00 0.31 0.00 0.00 23.3 NG
14 1 5.00 0.00 0.00 0.00 345.2 NG
2 2.50 0.00 0.00 0.00 172.6 NG
4 1.25 0.00 0.00 0.00 86.3 NG
8 0.63 0.00 0.00 0.00 43.2 NG
16 0.31 0.00 0.00 0.00 21.6 NG
15 2 0.00 0.00 0.00 2.50 79.7 PS
4 0.00 0.00 0.00 1.25 39.9 PS
8 0.00 0.00 0.00 0.63 19.9 NG
16 0.00 0.00 0.00 0.31 10.0 NG
Results summarised in Tables 10b and 10c showed that the batch LODs for
individual toxins were estimated to be below the regulatory limit for GTX2/3
and STX, with specific LODs of >19.9 µg STX eq./100 g for GTX2/3 and
>23.3 µg/100 g for STX. The LOD for NEO was however above the regulatory
limit with an estimated LOD of >86.0 µg STX eq./100 g for NEO. None of the
test strips inoculated with mussel extracts spiked with GTX1/4 at
concentrations up to 345.2 µg STX eq./100 g showed any positive response.
QC checks performed on mixtures of toxins in spiked mussel matrix showed
that although the batch was able to detect most samples containing various
profiles of toxins, it failed to detect one mixture (Mixture 1 – Table 10a)
containing toxins above the regulatory limit with a toxin profile consisting of
95% GTX1/4 and 5% STX. One of the 'real' toxin profiles observed on the NE
coast of England in 1990, also failed to be detected by JRT at the level of
fishery closure (Mixture 7 representative of a profile detected in mussels –
Table 10b). This sample profile consisted of NEO in isolation.
Finally, six of the fifteen spiked samples tested (40%) failed to be detected at
40 μg STX eq./100g), including one sample containing equimolar
concentrations of GTX1/4 and STX. These cases are highlighted in bold italic
in the above tables.
Page 16 of 314. Discussion and conclusion
The supporting literature for the Jellett Rapid Test for PSP states that the kit
will “detect all saxitoxins, the causative agent for Paralytic Shellfish Poisoning
(PSP) as low as 40µg/100grams” (Jellett Rapid Testing Ltd’s web site,
http://www.jellett.ca/psp_test.htm, 14/12/2006). A certificate of analysis (CoA)
is provided with test kit batches certifying the quality of the batch with regards
to response to STX and NEO. The average detection limit of the batch is also
given. Batch 40000-19Apr05 was stated in the CoA to have “an average
detection limit in naturally contaminated extracts of 40 μg FDA STX eq./100g”.
Batch 40000-19Apr05 was received by Cefas in February 06 having been
recertified by Jellett Rapid Testing Ltd until January 2007 (i.e. shelf life
extended by 8 months). The batch was submitted to additional internal quality
controls (as described in this report) to establish its response to all PSP toxins
since the information provided by the CoA related to NEO and STX only and
did not provide specific LODs.
Since a comprehensive suite of certified reference shellfish material
containing each of the PSP toxins is not available, the QC testing conducted
by Cefas used shellfish extracts shown to be negative for PSP toxins by MBA
(and JRT) and then spiked with known amounts of toxin standards. This
approach is widely used within analytical laboratories for either method
development or quality control of routine methods. The single toxin approach
is as recommended by the UKNRL for the performance of QC on the Jellett
Rapid Test for PSP and is used by the kit’s manufacturer as part of their
routine quality control system for the kit. Some of the data published by the
company, for example Laycock et al. (2001, 2004) [5,6] also relates to
sensitivity studies conducted on spiked shellfish extracts (single toxins or toxin
mixtures) or on solutions of pure toxins diluted in the running buffer.
The results of the QC evaluations showed that batch 40000-19Apr05 was able
to detect STX, dcSTX, GTX2/3 and GTX5 (B1) at concentrations well below
the regulatory limit of 80 μg STX eq./100g. However the batch was found to
have a poor sensitivity to NEO and GTX1/4. The kit’s response to GTX1/4 in
particular was very significantly in excess of the regulatory limit. The reactivity
established to NEO and GTX1/4 could not be reconciled with the
manufacturer’s claim that the kit can detect “all saxitoxins as low as
40µg/100grams”. Jellett Rapid Testing Ltd were contacted and they
subsequently provided data on two occasions on the specific limits of
detection of batch 40000-19Apr05. Comparison of Cefas and Jellett Rapid
Testing Ltd sets of data showed similar LODs were obtained for most of the
toxins. The new data from Jellett Rapid Testing Ltd for GTX1/4 confirmed the
variable sensitivity of the kit for this toxin. LODs for NEO and GTX1/4 found in
this study suggested a reduced level of sensitivity of the kit for these toxins
compared with that previously reported by the manufacturer [5,6].
There are few published papers describing the response of the JRT kit to
specific PSP toxins. Most of the other data available on the JRT relates to its
performance with naturally contaminated field samples. Laycock et al. (2001,
Page 17 of 312004) described the characteristics of the PSP test and its relative sensitivities
to a range of purified PSP toxins. All toxins were shown by the authors to be
detectable at below the regulatory limit but they also showed that the
sensitivity to NEO and GTX1/4 were about five fold less than to STX and its
analogues. LODs for NEO and GTX1/4 could not be established from data
presented in Laycock et al. (2004). However, they could be calculated from
data provided in June 2006 to the UKNRL by Maurice Laycock (Chief
Scientist, Jellett Rapid Testing Ltd) and were calculated as 49 and 55 μg STX
eq./100g for NEO and GTX1/4, respectively. Thus the comparatively weaker
response of the JRT kit to NEO and GTX1/4 compared with, for example, STX
is documented.
However, though in 2004 the LODs reported were still below the regulatory
level, the QC study for batch 40000-19Apr05 demonstrated an increase of the
LODs by a factor of 3 for NEO and a factor of 22 for GTX1,4 compared to the
initial studies. Data subsequently obtained from Jellett Rapid Testing Ltd for
more recent batches of the PSP JRT kit suggest that batch 40000-19Apr05
was not abnormal as similar responses were obtained for these batches
(batches 16Feb06 and 21Jul06 – data not shown).
QC checks conducted on material spiked with mixtures of PSP toxins showed
that batch 40000-19Apr05 failed to detect several samples containing toxin
mixtures at levels above the stated kit and batch LOD of 40 μg/100g. In
addition to the limitations of the batch with regard to GTX1/4 alone tests
containing approximately 40 μg/100g of PSP toxins made up of GTX1/4 in
combination with STX were also not detected, both when GTX1/4 was
predominant but also at the ratio of 50:50. Two test samples were not
detected by 40000-19Apr05 at the regulatory limit. One test contained 95%
GTX1/4 and 5% STX and the second, representative of a toxin profile seen in
a mussel sample during a large PSP incident in 1990, consisted of NEO in
isolation.
It has been suggested that the JRT performs better in the presence of toxin
mixtures. During the QC work, no evidence was seen for enhancement of
overall test sensitivity to each toxin in the presence of toxin mixtures. For
example, the test did not detect 1.25 μM of GTX1/4 in isolation or 1.19 μM
GTX1/4 in the presence of 0.06 μM of STX. The possibility that the JRT
performs better in the presence of toxin mixtures compared to individual toxins
would need to be demonstrated with additional experimental work as thus far
there is insufficient evidence to support it.
The results reported here raise concerns over the reactivity of JRT batch
40000-19Apr05 in relation to two individual PSP toxins and test some possible
implications of this reactivity spectrum for the detection of PSP toxins in real
life samples. The results suggest that use of JRT batch 40000-19Apr05 poses
a risk for non-detection of certain combinations of PSP toxin in samples at the
regulatory limit. The extent of the problem will depend on the toxin profiles in
individual samples and comprehensive profiling data is required to establish
the risk.
Page 18 of 315. Consequential actions
Following discussion of the findings documented in this report the UKNRL
issued a recommendation dated 25th May 2006 on the use of JRT in the UK
statutory PSP monitoring programme which advised suspension of the use of
batch 40000-19Apr05. This was based on the data presented in section 3.1.
The UKNRL subsequently issued a SOP detailing the QC performance criteria
checks to be conducted prior to use of the Jellett Rapid Test for PSP in official
monitoring laboratories, based on the JRT available literature from 2001 and
2004 [5,6] and the regulatory limit. Batches 40000-19Apr05 and subsequent
JRT kit batches (16Feb06 and 21Jul06) received by Cefas have not complied
with the requirement laid out in the UKNRL SOP for JRT QC and have
therefore not been used for official control testing for PSP in the UK.
Page 19 of 31PART TWO
Analysis of field samples for paralytic shellfish
poisoning toxins by the Jellett Rapid Test
26th May to 31st October 2006
Page 20 of 311. Introduction and background
Paralytic shellfish poisoning (PSP) toxins are potent neurotoxins produced by
certain marine dinoflagellates and can accumulate in filter feeding molluscan
shellfish to highly toxic levels. Shellfish destined for human consumption are
required to be routinely monitored to protect consumer’s health [1].
In the UK, the analysis for PSP toxins of shellfish collected under the scope of
the Official Control (OC) national biotoxin monitoring programme is based on
the biological assay (MBA). The MBA is an AOAC (Association of Official
Analytical Chemists) Official Method (method 959.08) [2] and is also the
reference method specified in European legislation on seafood safety (EC
Regulation 1664/2004 [7]). Recent improvements in analytical techniques for
PSP testing present opportunities to explore alternatives to the MBA for OC
testing.
In 2005 it was agreed to initiate a programme of evaluation for the use of High
Performance Liquid Chromatography (HPLC) method with fluorescence
detection and pre-column oxidation as an alternative to the MBA for PSP
detection. The method was adapted from that described by Lawrence et al.
[8,9,10,11,12,13] for the determination of saxitoxin (STX), gonyautoxins (GTX)
2,3 together, 1,4 together and 5 (also known as B1), neosaxitoxin (NEO),
decarbamoyl saxitoxin (dcSTX) and C1,2 together and C3,4 together in
mussels, clams, oysters and scallops [14]. This method was agreed by AOAC
International in June 2005 as an Official First Action Method (method 2005.06)
for determination of certain PSP toxins in some shellfish species. The
modifications to the methodology related to its use, in the first instance, as a
qualitative screen. The performance of the HPLC screen was assessed
through a single laboratory validation exercise against molluscan shellfish
species of commercial relevance to the UK and for all PSP toxins for which
certified reference standards were available.
Following this the HPLC screen was evaluated with field samples for a 5
month period. During that period action was taken on MBA results, performed
in parallel with the HPLC. The results of these evaluations will be reported
elsewhere. As part of this programme a commercial PSP toxins screening
assay, the Jellet Rapid Test (JRT) was also incorporated into the trial. This
report describes the experiences gained with the JRT and the results obtained
from 26th May to 31st October 2006.
The JRT is a commercial antibody-based qualitative test manufactured by
Jellet Rapid Testing Ltd, Canada. The test uses polyclonal PSP toxin
antibodies and works on the principle of lateral flow immuno-chromatography
using a strip format (similar to pregnancy kits). The test is stated by the
manufacturer to detect PSP toxins at above 40 µg/100g (Jellett Rapid Testing
Ltd’s web site, http://www.jellett.ca/psp_test.htm, 14/12/2006) and is marketed
as a qualitative screen assay.
Page 21 of 31For JRT analysis shellfish extracts are diluted in JRT running buffer, loaded
onto a JRT detection strip and incubated for 35 minutes and the test result
evaluated by monitoring a colour reaction in the test and control strips.
Following incubation the colour intensity of the T line is read against that of
the C line to define whether the sample is positive or negative. The schematic
below illustrates positive and negative tests.
C-line
T-line
Positive sample
Negative sample
Following previous field evaluations reported elsewhere the JRT kit (formerly,
MIST Alert) was used for some shellfish species, as a qualitative screening
test for PSP toxins in the Scottish biotoxin monitoring programme. However,
its use was suspended in May 2006 following concerns about the spectrum of
response to individual PSP toxins (See Part One of this report). The JRT is
not currently used as a component of the UK official control monitoring
programme for marine biotoxins.
2. Materials & methods
Shellfish samples
961 shellfish samples, submitted to the laboratory for PSP toxin analysis
between 26th May and 31st October 2006, were included in the trial. These
samples consisted of Common mussels (Mytilus edulis) (640), Pacific oysters
(Crassostrea gigas) (142), Native oysters (Ostrea edulis) (101), Queen
scallops (Aequipecten opercularis) (17), Razor clams (Ensis sp.) (6), Common
cockles (Cerastoderma edule) (51), Hard clams (Mercenaria mercenaria) (3)
and King scallops (Pecten maximus) (1), originating from the Scottish,
English, Welsh, Isle of Man and Isle of Jersey biotoxin statutory monitoring
programmes.
Page 22 of 31Jellett Rapid Test batches
Strips used in the trial were from batches 40000-24Feb05, 19Apr05, 16Feb06
& 21Jul06, purchased from Jellett Rapid Testing Ltd, Canada. Buffer
reference 40004-04Jan06, 40004-12Jul05, 40004-07Jun06 and 40004-
08Aug06 were supplied by the manufacturer with the above batches of strips.
JRT kits were provided with Certificate of Analysis (CoA) stating that they had
passed the manufacturer’s quality controls (QC) and that the response of the
kit to “several concentrations of STX and NEO” was “consistent with the
previously released lot of the same type”. Batches 40000-24Feb05 and
19Apr05 had been recertified by the manufacturer and the shelf-life of both
batches extended until Jan07. All CoAs stated that the above batches had “an
average detection limit in naturally contaminated extracts of 40 μg FDA STX
eq./100g”.
The UKNRL SOP on QC procedures to be used for the Jellet rapid test for
PSP in monitoring laboratories, stipulates that for a PSP JRT batch to be
accepted for use within the monitoring programme QC data must be supplied
by the manufacturer for a range of relevant toxins. Information from each
batch should show that it will detect the specified toxins at the LODs defined
by the SOP for these toxins. The SOP LODs were based on data published by
Laycock et al. (2004) [6] and supplied by Maurice Laycock (Chief Scientist,
Jellett Rapid Testing Ltd) to the UKNRL in June 2006.
On request from Cefas, data on the limits of detection (LOD) for individual
PSP toxins of the four batches used during the parallel trial was supplied by
Jellett Rapid Testing Ltd (confidential data). The data provided by Jellett
Rapid Testing Ltd was not always complete with regards to the spectrum of
toxins specified by the UKNRL QC SOP. The stated response of the batches
to NEO and GTX1/4 exceeded the required LOD specified in the UKNRL QC
for these toxins (49 and 55 μg STX eq./100g, respectively). Moreover, the
LOD stated by the manufacturer for GTX1/4 was significantly above the
regulatory limit of 80 μg STX eq./100g. Internal QC checks performed on
batch 40000-19Apr05 confirmed that this batch did not meet the UKNRL QC
SOP specifications, as the LODs for NEO and GTX1/4 were estimated to be
>160 and >1,216 µg STX eq./100g respectively (See Part One of this report).
The JRT batches used in this study therefore did not comply with the quality
assurance criteria specified in the UK NRL QC SOP for use in official control
testing. JRT test results reported here were not used for official control
purposes.
Tissue extraction and MBA testing
All samples were extracted for testing by MBA in accordance with Cefas SOP
on PSP extraction. All extracts were tested by MBA within 24h of extraction in
accordance with Cefas SOP. Where necessary HCl extracts were stored at
+4ºC prior to testing. Cefas procedures for extraction and testing for PSP by
MBA are based on the AOAC method 959.08 and the test method is
accredited by UKAS under ISO 17025.
Page 23 of 31JRT assay
Testing of samples by JRT was performed in accordance with the UKNRL
SOP for the analysis of shellfish extracts by the Jellett Rapid Test and in
compliance with the manufacturer’s instructions. JRT testing was performed
immediately after sample extraction. Using a calibrated pipette 400 μl of JRT
running buffer was placed into individual microcentrifuge tubes, 100 μl of test
sample was then added and mixed with buffer by pipetting. 100 μl of the
mixture was then placed onto the sample pad of the JRT detection strip and a
timer starter. Strips were incubated at room temperature for 35 minutes and
the colour reaction documented. Results were interpreted according to the
Jellett Rapid Test Instruction Sheet. Strips were read by a trained analyst and
the test result checked by a second analyst.
In accordance with the manufacturer’s instructions received with batches
40000-24Feb05, 19Apr05 and 16Feb06, a result was recorded as positive
when the intensity of the T line was 0%, 25% or 50% of that of the C line, and
negative when the intensity of the T line is 75% or 100% of that of the C line.
In accordance with the manufacturer’s instructions JRT results were
considered invalid if the C-line intensity was equivalent to or less than 25% of
that on the supplied demonstration unit or if a T line was observed with no
apparent C line. In this case the test was repeated and the result of the
subsequent valid test recorded for the affected sample.
On 22nd August 2006 Jellett Rapid Testing Ltd issued a revised instruction
sheet for batch 40000-16Feb06. This was issued to address the problem of
subjectivity over the interpretation of positive and negative results for this
batch and to clarify what constituted an invalid result. A result was considered
positive if the sample T line was equal to, or less than, the intensity of an
illustrative T line shown on the instruction sheet and negative if the sample T
line was darker than the illustrative positive T line. An invalid test was a test
showing a C line with an intensity equal to or fainter than that of the illustrative
invalid test shown on the instruction sheet. Similar instructions were supplied
with batch 40000-21Jul06.
To maintain consistency during this field trial the interpretation of test result for
batch 40000-16Feb06 remained according to the manufacturer’s original
instructions for that batch. However, a second reading was performed using
the revised instructions on completion of the trial. This was performed on all
697 samples tested using batch 40000-16Feb06 between 21st June and 27th
September 2006. All test strips were stored in sealed pouches and in the dark
prior to reanalysis.
Strips from batch 40000-21Jul06 were read according to the instructions
received with the batch.
Page 24 of 313. Results 3.1. Invalid tests Using the manufacturer’s original instructions a total of 8 tests (
Table 3: Summary of agreement in the interpretation (T line response) of JRT results between four
independent analysts (A-D)
Concentration Proportion agreement (%)
NEO
Replicate 1 Replicate 2 Replicate 3
2AL 75 75 50
1AL 100 75 50
1/2AL 100 100 100
C-line
T-line
C-line
T-line
50% 25%
50% 75%
50% 75%
Figure 1: Jellett strips inoculated in triplicate with mussel extract spiked with Neosaxitoxin at twice the
regulatory limit (on the left) and at the regulatory limit (on the right) – Interpretation of Analyst A
Results showed that all test strips inoculated with NEO at ½ AL yielded
negative results and all analysts read the test results as negative (100%
agreement).
At 1AL, only one of the inoculated strips yielded a positive result which was
consistently scored as positive by the four analysts. The other two strips
inoculated with NEO at 1AL returned a negative result. All analysts scored
these strips as negative but interpreted T lines intensities of both strips as
either 75 or 100% of the C line intensity.
At 2AL, one of the strips was consistently scored positive by all four analysts
but their T line interpretation varied from 50 to 25% of the C line intensity. The
other two strips inoculated with NEO at 2AL were read positive by 3 (Strip 2)
or 2 (strip 3) analysts.
Therefore overall, although seven of the nine inoculated strips were
consistently read negative or positive by all analysts, only four of the nine
strips were given the same T line interpretation by all four analysts. The T
lines of these four strips were either all scored as 25%(1 strip) or 100% (3
Page 26 of 31strips) of the C line intensity. Strips yielding an intermediate response yielded
the most variation in interpretation of their T line intensity.
These results illustrate the subjective nature of result interpretation particular
around the 50% intensity end-point of the assay and, consequently, the
variability observed in analyst interpretation (Tables 1 to 3).The
manufacturer’s revised set of instructions for batch 40000-16Feb06 partially
address interpretation subjectivity by scoring line intensity against a supplied
positive T line image. However comparative evaluation of C and T line
intensity is still required and thus the assay remains susceptible to variation in
analyst scoring.
Complications were also experienced with faint C-lines and the variability of
C-line response within a batch. These resulted in difficulty in interpreting the
relative intensity of the T line and therefore the positivity of the sample. A
number of the tests performed during the reported period showed C lines of
lower intensity than the test line. Figure 2 below (strips 633 and 640S (2))
illustrates this issue.
Figure 2: Jellett strips inoculated with shellfish samples 633 and 640S(2) showing faint C-lines
The manufacturer recommends that sample showing doubtful results should
be tested using an alternative method. The UKNRL SOP also recommends
that if there is any doubt about a result, or if a strip is difficult to read, the
sample should be tested by MBA. This approach may help reduce the risk of
false negative results due to subjectivity in test result interpretation.
3.3. Results of JRT with field samples
All 961 shellfish field samples were tested by both MBA and JRT. For the
purposes of this report an MBA positive sample was defined as one that had
Page 27 of 31detectable levels of PSP by this assay. Of the 961 samples tested 65 were
MBA positive; all of these samples were also found positive by JRT. Of the
896 remaining samples (all MBA negative), 36 were found positive by JRT. All
860 samples which were found to be JRT negative were also MBA negative.
Table 4 below summarises the results obtained in each shellfish species.
Table 4: Summary of JRT and MBA results obtained in each shellfish species tested
Shellfish species No. samples JRT results MBA result
tested Negative Positive Not detected Positive
Hard clams 3 3 0 3 0
Razor clams 6 2 4 3 3
Common cockles 51 51 0 51 0
King scallops 1 1 0 1 0
Queen scallops 17 17 0 17 0
Pacific oysters 142 139 3 142 0
Native oysters 101 100 1 101 0
Common mussels 640 547 93 578 62
As indicated in Table 4, 62 of the 65 samples (95.4%) found in this study MBA
positive were common mussel samples. The rest were razor clam samples.
All cockles, scallops or oysters tested were found negative by MBA.
All three razor clams and 56 of the 62 mussel samples found MBA positive
originated from Scotland (data not shown).
The 36 samples found JRT positive but MBA negative consisted of mainly of
common mussels (29 samples, i.e. 4.5% of all mussel samples) but also razor
clams (1), pacific oysters (3) and native oysters (1).
The rescoring of results of the JRT strips of batch 40000-16Feb06 using the
manufacturer’s revised instructions showed that the revised assessment only
affected three test results: two negative samples were rescored as positive
and one positive sample was rescored as negative. All three samples were
MBA negative.
4. Discussion and Conclusion
The JRT evaluations reported in this study were performed during a
comprehensive evaluation of an HPLC screen for the detection of PSP toxins
included the use of the JRT. The evaluation of the HPLC screen is reported
elsewhere (report in preparation). The JRT batches supplied by the
manufacturer for use in this study were found not to comply with the quality
assurance criteria specified by the UK National Reference Laboratory and the
LODs for neosaxitoxin and gonyautoxins 1 and 4 were above the regulatory
limit. Therefore these batches could not have been used for the official control
monitoring programme.
The subjective nature of the assay scoring was found to complicate result
interpretation and could lead to variable interpretation particularly around the
50% end-point of the assay. In a short study aimed at investigating variability
in analyst interpretation, seven of the nine (78%) inoculated strips were
Page 28 of 31consistently read negative or positive by all analysts. However only four of the nine strips (44%) were given the same T line interpretation by all four analysts. These strips all showed strong positive (T line intensity of 25%) or negative (T line intensity of 100%) response. These results are similar to those reported by Mackintosh et al. (2002) [15] who assessed the reproducibility and ease of interpretation of the MIST Alert, a previous version of the JRT. In their study, ten JRT strips were independently scored by seven novices and one expert. Eight of the 10 strips (80%) were consistently scored either positive or negative by the analysts. As above, agreement in T line interpretation was obtained for all samples (6 out of 10) showing either a strong positive or negative response. The four samples which yielded some variation in T line interpretation were scored as either 50 or 100% by the expert but scored as 25, 50 or 100% by the novices. The new set of instructions released by the manufacturer may help reduce subjectivity in scoring test results. Better results may however be obtained by automation of the reading process. A proportion of the strips used between May and October 2006 yielded invalid results. Overall,
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