WAVLD Symposium Handbook_V4.indd - csiro

World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
THE DEVELOPMENT OF SNP DISCRIMINATION ASSAYS ON A REAL-TIME PCR PLATFORM AS A
TOOL FOR THE RAPID IDENTIFICATION AND SPECIATION OF BRUCELLA.
K.K. Gopaul, C.J. Smith and A.M. Whatmore*
Department of Statutory and Exotic Bacterial Disease, Veterinary Laboratories Agency, Woodham Lane, Addlestone, Surrey, United
Kingdom, KT15 3NB. [a.whatmore@vla.defra.gsi.gov.uk]
Introduction. Brucellosis, caused by members of the genus Brucella, remains a major zoonotic problem in
much of the world. Six distinct species are identified within the genus; B. abortus (bovine), B. melitensis
(caprine, ovine), B. suis (porcine, rangiferine, leporine), B. canis (canine), B. ovis (ovine) and B. neotomae
(desert wood rat) with evidence of additional species, yet to be formally named, associated with various
marine mammal species [1]. Currently the most widely recognised tool for Brucella speciation is biotyping but
this method is slow, can be very subjective and involves hazardous culture of a pathogen readily acquired by
laboratory workers. The aim of this work is therefore to develop a rapid molecular alternative that can initially
identify to species level but will ultimately be extended, if possible, to include markers for subgroups (e.g.
biovars) and live vaccine strains.
Materials & Methods. Single nucleotide polymorphisms (SNPs) specific for each Brucella species were
identified on the basis of an extension of multilocus sequencing studies previously described by us [2]. Some
21 distinct genomic fragments, representing >10 Kb of sequence, were sequenced from >300 Brucella
isolates representing the known diversity of the group. Based on this work, that comprises the most
extensive study of the population structure of the genus undertaken to date, pairs of MGB allele
discrimination probes were designed and tested. Probes corresponded to species-specific SNPs and the
alternative ‘common’ allelic state, and were designed for use in real-time PCR reactions to discriminate each
of the six Brucella species and the marine mammal Brucella.
Results. The performance of various probe pairs designed to interrogate SNP sites with a change specific
for each of the individual species was assessed and those that provided most promising discrimination were
selected. Probe pairs were all optimised to function under identical PCR conditions allowing development of
a multiplex assay. Once optimised the performance of the resulting ‘speciation’ assay was assessed by
testing over 300 field and reference isolates of Brucella. Sensitivity of the assay was determined by testing
serial dilutions of purified genomic DNA while specificity was assessed by testing organisms phylogenetically
related to Brucella.
Discussion and Conclusions. Use of multilocus sequence data from several hundred strains facilitated the
confident design of a diagnostic assay based on a robust phylogenetic framework. The assay can ‘speciate’
an isolate in around 2 hours and has many advantages over biotyping being much easier to perform, less
labour intensive, less subjective and avoiding extensive culturing of this hazardous agent. Furthermore, the
principle of the assay can now be extended to further subtype beyond the species level by incorporating
additional markers to identify subgroups, such as biovars, and live vaccine strains. The assay will also be
amenable to future expansion should additional Brucella species be identified – suitable markers for
inclusion should readily be identified by undertaking the same multilocus sequencing approach.
References.
[1] Groussaud, P., Shankster, S.J., Koylass, M.S., and Whatmore, A.M. 2007. Molecular typing divides
marine mammal strains of Brucella into at least three groups with distinct host preferences. Journal of
Medical Microbiology. In press.
[2] Whatmore, A.M., Perrett, L.L., and Macmillan, A.P. 2007. Characterisation of the genetic diversity of
Brucella by multilocus sequencing. BMC Microbiology 7:34.
Mon 12 November
Mon 12 November
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
DETECTION OF MYCOPLASMA HYOPNEUMONIAE IN PIG SAMPLES
USING POLYMERASE CHAIN REACTION TESTS
G. J. Eamens, NSW Department of Primary Industries
Introduction
Mycoplasma hyopneumoniae (Mhp) is a major respiratory pathogen of pigs with worldwide distribution. It is
responsible for lost production through its ability to induce pneumonia but particularly to predispose pigs to
secondary respiratory infections with other bacterial and viral pathogens. As Mhp is difficult to culture from
field material, diagnostic testing has relied on serological and PCR-based assays to indicate herd infection.
Numerous reports describe PCR tests for the specific and sensitive detection of Mhp, varying from
conventional one step and two step (nested) assays to real time PCR, and targeting numerous DNA
sequences considered specific for Mhp. Few have been examined concurrently, and thus diagnostic
laboratories tend to rely on data collected with limited head-to-head evaluation.
Material & methods
This study reviewed 25 PCR assays reported in the literature since 1993. It targeted conventional PCR
assays reported with sufficient information to score the features of each. The factors considered of interest
were: sensitivity < 10 fg, prior evaluation in lung tissue, nasal swabs and other tissues, as well as a proven
ability to detect M. hyopneumoniae in nasal swabs, and prior evaluation with commercial extraction kits. In
the initial phase, issues affecting test evaluation in “beta testing” assays were identified. Cultures of Mhp and
related mycoplasmas (M. flocculare, M. hyorhinis) were also obtained to assist in test optimisation prior to
application to field samples from several herds with high and low disease status for mycoplasmal pneumonia
(nasal swabs from weaner and finisher pigs, lung samples from affected slaughter pigs).
Results and discussion
Among nine conventional PCR assays which were well described and for which analytical sensitivity and
application data was available, five assays with reported high sensitivity were able to be selected for
concurrent evaluation (Table 1). Among nine commercial DNA capture kits applied to M. hyopneumoniae
PCR tests on tissues, two currently used in Australian diagnostic laboratories (Instagene matrix, BioRad;
DNeasy, Qiagen) were selected for comparative evaluation. Optimisation studies using DNA from previously
characterised mycoplasmal strains showed some published protocols required adjustment of annealing
temperatures and some assays showed cross-reactivity or failure to detect some Mhp strains. Efforts to
clone some cultured strains and apply multiplex assays were also required to validate the source strains.
Table 1. Comparative literature evaluation of conventional PCR for M. hyopneumoniae
Type Test Analytical
sensitivity
PCR
nested
PCR
Artiushin 1 1-10 pg 1 ;1-10 pg 7
Mattsson 8
6 fg 8 ; 100 fg 7
Blanchard 3
0.5 pg 3 ; 10 pg 7
Baumeister 2 5-18.5 fg 5
Caron 6 50 pg 6 ; 1 pg 7
Stark 9 1.2 fg 9 ; 2.5 fg 7
Calsamiglia 4 96 fg 4 ; 1 fg 7
Verdin 10 1 fg 10 , 1 fg 7
Kurth 7 0.5 – 1 fg 7
High
sensitivity
< 10 fg
Tested
on lung
tissue
Tested on
nasal
swabs
Tested
on other
tissue
Detects in
nasal
swabs
DNA kit
used
- - - - - -
- + + + + -
- - - + - -
+/- + + + - +
- + - + - +
+ + + + + +
+ + + + + +
+ + - + - -
+ + + + + +
Conclusions
A significant investment can be required in the laboratory introduction of diagnostic Mhp PCR, and may
require extensive in-house validation to optimise the assay and confirm specificity.
References
1. Artiushin S, Stipkovits L, Minion FC (1993). Molec Cel Probes 7, 381-385.
2. Baumeister AK, et al H (1998) J Clinical Microbiol 36, 1984-1988.
3. Blanchard B, et al (1996). Molec Cell Probes 10, 15-22.
4. Calsamiglia M, Pijoan C, Trigo A (1999). J Vet Diag Invest 11, 246-251.
5. Carew D (2004). PhD thesis, Swinburne Univ of Technology 56-63; 97-111.
6. Caron J, Ouardani M, Dea S (2000). J Clin Microbiol 38, 1390-1396.
7. Kurth KT, et al (2002). J Vet Diag Invest 14, 463-469.
8. Mattsson JG, et al (1995). J Clin Microbiol 33, 893-897.
9. Stark KDC, Nicolet J, Frey J (1998). Appl Environ Microbiol 64, 543-548.
10. Verdin E, et al (2000). Vet Microbiol 76, 31-40.
Supported by Australian Pork Limited