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EVALUATION OF A NESTED PCR ASSAY FOR
IDENTIFICATION OF VIRULENT Rhodococcus equi
M.L. MarenzoniM.L. Marenzoni11, F. Passamonti, F. Passamonti11, K. Cappelli, K. Cappelli11, S. , S. Capomaccio Capomaccio 22, F. Cittadini, F. Cittadini11, M. Catanossi, M. Catanossi11, G. , G.
CoppolaCoppola11, M. Coletti, M. Coletti11..Centro di Studio del Cavallo Sportivo Centro di Studio del Cavallo Sportivo
11Facoltà di Medicina Veterinaria, PerugiaFacoltà di Medicina Veterinaria, Perugia 22Facoltà di Agraria, PerugiaFacoltà 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
Primer 16S-forward 5’-TCGTCCGTGAAAACTTGGG-3’
Primer 16S-reverse 5’-ACCACAAGGGGGCCGT-3’
5’-GAGGAGCGAAAGCGTGGGTA-3’Primer 16S N-reversePrimer 16S N-forward
5’-TTAGCCTTGCGGCCGTACTC-3’
*Sellon D.C., et al., 2001
Access Genbank X82052
Materials and Methods: PCR primers for plasmid VapA of Rhodococcus equi
Primer VP N-forward 5’-TCGGAACTGCCCGAGAACAT-3’Primer VP N-reverse 5’-GCTCCCAGAACCGACAATGC-3’
5’-GAGGGATCCGGTTCTCGTAACGCTACAATC-3’Primer VP-reversePrimer VP-forward
5’-GGTTCGTCTTTCTGAAGGTT-3’*Sellon D.C., et al., 2001
Access Genbank AF116907
94°C
66°C
68°C30 ‘‘
30 ’
1 ’
35 cicli94°C
1 ’
*Sellon D.C., et al., 2001
94°C
62°C
68°C30 ‘‘
30 ’
1 ’
35 cicli94°C
1 ’
THERMAL CYCLE: FIRST ROUND
THERMAL CYCLE: NESTED ROUND
MATERIALS AND METHODS
• 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: specimens
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
16S first round VP first round
16S nested
M 0,2ng 0.02ng 2pg 0.2pg N BC+DNA R.equi N LINF+DNA R.equi
N: negative control;BC: DNA buffy coat; LINF:DNA lymph node.
VP nested
M 0,2ng 0.02ng 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 in retrospective studies or environmental investigation
Grazie per l’attenzione!
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