Download Evaluation of a nested PCR assay for identification of virulent

EVALUATION OF A NESTED
PCR ASSAY FOR
IDENTIFICATION OF VIRULENT
Rhodococcus equi
M.L. Marenzoni1, F. Passamonti1, K. Cappelli1, S.
Capomaccio 2, F. Cittadini1, M. Catanossi1, G.
Coppola1, M. Coletti1.
Centro di Studio del Cavallo Sportivo
1Facoltà di Medicina Veterinaria, Perugia
2Facoltà di Agraria, Perugia
INTRODUCTION
•
Rhodococcus equi is a Gram-positive bacteria and an opportunistic pathogen of
foals under 6 months of age
•
R equi is an inhabitant of both soil and intestinal tracts of animals
•
Suppurative bronchopneumonia of foals is the major disease caused
•
Pigs, cats and cattle can occasionally be infected
•
Pneumonia caused by R. equi has been reported in patients with human
immunodeficiency virus infection
•
Only virulent strains of R. equi harbouring a virulence plasmid of 85 to 90 kb
(VapA)
•
The disease tends to be insidious and lesions can be well advanced before the
animal exhibits coughing, dyspnoea,and characteristic loud, moist rales on
auscultation of the lung
•
Attempted culture of R. equi is often unrewarding
OBJECTIVE
•
To develop a new nested PCR protocol with the aim to increase sensitivity
and specificity to detect R. equi and to differentiate strains that contain the
virulence-associated gene (VapA) from strains that do not
Materials and Methods: PCR primers for
16S rRNA gene of Rhodococcus equi
Access Genbank X82052
Primer 16S-forward
Primer 16S-reverse
5’-TCGTCCGTGAAAACTTGGG-3’
5’-ACCACAAGGGGGCCGT-3’
*Sellon D.C., et al., 2001
Primer 16S N-forward
Primer 16S N-reverse
5’-GAGGAGCGAAAGCGTGGGTA-3’
5’-TTAGCCTTGCGGCCGTACTC-3’
Materials and Methods: PCR primers for
plasmid VapA of Rhodococcus equi
Access Genbank AF116907
Primer VP-forward
Primer VP-reverse
5’-GAGGGATCCGGTTCTCGTAACGCTACAATC-3’
5’-GGTTCGTCTTTCTGAAGGTT-3’
*Sellon D.C., et al., 2001
Primer VP N-forward
Primer VP N-reverse
5’-TCGGAACTGCCCGAGAACAT-3’
5’-GCTCCCAGAACCGACAATGC-3’
MATERIALS AND METHODS
THERMAL CYCLE: FIRST ROUND
94°C
35 cicli
68°C
30 ‘‘
1’
62°C
30
’
THERMAL CYCLE: NESTED ROUND
94°C
35 cicli
68°C
30 ‘‘
1’
66°C
30
*Sellon D.C., et al., 2001
’
MATERIALS AND METHODS: specimens
•
To evaluate sensitivity serial tenfold diluitions from 20 ng to 0,2 fg.
of DNA from R. equi reference strain (ATCC 33701) were carried out
•
Twenty-four R. equi isolates (obtained from colonies on blood agar and
identified by biochemical test -Api Coryne, Biomérieux- and CAMP test)
•
Ten biological specimens from horses with clinical disease (ie, tracheal wash,
abscesses, lung tissues from foals with pneumonia)
•
To evaluate specificity Corynebacterium pseudotuberculosis, Escherichia coli,
Klebsiella pneumoniae, Nocardia asteroides, Pasteurella spp., Staphylococcus
aureus, Streptococcus equi, Mycobacterium spp. were tested with nested-PCR
MATERIALS AND METHODS: DNA
EXTRACTION
• R. equi isolates: 95 °C for 5’, centrifuged at 14.000rpm
for 5’  2 µl for PCR.
• Biological specimens: extraction with the QIAamp DNA
Blood Mini Kit  200 ng or 5 µl for PCR.
• DNA concentration and purity were measured with
spectrophotometer and electrophoresis on agarose gel
MATERIALS and METHODS: PCR
FIRST ROUND PCR:
R. equi isolates: 2 µl supernatant
• BIOLOGICAL SPECIMENS: 200 ng or 5 µl
NESTED CYCLE:
•
1 µl di of the first round product
RESULTS: sensitivity and specificity
• Sensitivity: 0,2 ng for the first round and 2 pg for the
nested round, for both genes (equivalent to 100 bacilli).
Sensitivity is 100 fold higher than the first cycle
• Specificity: no amplification occurred when different
bacteria from R. equi were used
VP first round
16S first round
M 0,2ng 0.02ng 2pg 0.2pg N BC+DNA R.equi
16S nested
N: negative control;
BC: DNA buffy coat;
LINF:DNA lymph node.
N
LINF+DNA R.equi
M 0,2ng 0.02ng
VP nested
BC+DNA R.equi
N
LINF+DNA R.equi
RESULTS: specimens
PCR R.equi isolates (n = 24):
•
All the isolates resulted positive for 16S and VapA genes in the first and in the
nested cycles; however the DNA in 6 specimens, extracted by boiling and stored
at -20°C for 3-4 years, resulted positive for the VapA gene only in the nested
cycle
PCR biological specimens
•
16S gene: all 10 specimens resulted positive both in the first and in the nested
cycle, with products of amplification more evident after the nested cycle
•
VapA : 8 resulted positive both in the first and in the nested cycle, whereas 2 (1
tracheal wash and 1 pulmonary nodul) only after the nested cycle
CONCLUSIONS
• Rapid identification of species and virulence plasmid, both in
contaminated samples and directly from specimens
• Increase in analytical sensitivity and specificity, especially in
clinical samples and in the identification of the virulence plasmid
• Possible use
investigation
in
retrospective
studies
or
environmental
Grazie per
l’attenzione!