New Method For Molecular Diagnostics In Veterinary Medicine Dr. Mikael Leijon
The challenge in genetic detection the third position is often undetermined but variation also exist in the first (L, S, R) and second (S) position. Conventional genetic detection methods requires conserved regions where primers and probes can bind. U C G U F F L L S S S S Y Y Stop Stop C C Stop W U C G C L L L L P P P P H H Q Q R R R R U C G I I I M T T T T N N K K S S R R U C G G V V V V D D E E G G G G U C G 1st position 2nd position 3rd position T h e d i l e m m a o f m o l e c u l a r d i a g n o s t i c s
E x e m p e l N e w c a s t l e D i s e a s e V i r u s ( N D V ) NDV whole genome NDV conserved region Blue color 100% sequence conservation
E x e m p e l N e w c a s t l e D i s e a s e V i r u s ( N D V ) This example illustrates typical sequence variation of an RN-virus. This is one of the best conserved regions of the NDV genome (nucleotides labeled blue are conserved). Despite this more than a third sequence positions are not conserved (no color). The periodic variation every third position is clearly visible. NDV conserved region
N o v e l G e n e r a l D e t e c t i o n C o n c e p t HIGH CONC (µm) PRE-MPLIFICTION GENERL DETECTION WITH HIGH SENSITIVITY Barrier against cross-contamination LOW CONC (nm) SPECIFIC MULTIPLEX RECOGNITION/TGING HIGH SPECIFICITY/ DPTBILITY HIGH CONC (µm) GENERIC MPLIFICTION/DETECTION TG-BSED DETECTION/ MPLIFICTION
S c h e m a t i c v i e w o f t h e m e t h o d Conventional PCR The new principle Microorganism genome Primer Multiplex Primer cocktail that recognize all sequence variants Pre-amplified microorganism genome
I p r a c t i c e P a t h o t y p n i n g o f a v i a n i n f l u e n z a Hemagglutinin pro-protein Enhanced cleavability result if the cleavage site has the motif : R-X-K/R-R Systemic infection with high pathogenicity H0 R-X-K/R-R Cleavagesite H1 H2
I m p l e m e n t a t i o n f o r I V - p a t h o t y p n i n g PCR 2 The new system PCR 1 primer primer primer probe primer Conventional system Cleavage site primer
I m p l e m e n t a t i o n f o r I V - p a t h o t y p n i n g Selection primers: fshp01 CGGGCTTCCCCCCCCCGCGGGRGRGGG fslp14 CGGGCCCCCTTCCCCCGTYCCTCRRGRCRGG fshp02 CGGGCTTCCCCCCCCCGGGGGGGGGGG fslp18 CGGGCCCCCTTCCCCCGTYCCTCRRGRCRGG fshp03 CGGGCTTCCCCCCCCCGCCCTCGGCGG fslp19 CGGGCCCCCTTCCCCCGGTYCCTCRRGGCRGG fshp28 CGGGCTTCCCCCCCCCGCGGRGGRRGRG fslp21 CGGGCCCCCTTCCCCCGGTYCCTCRRGRTCRGG fshp29 CGGGCTTCCCCCCCCCGGGRGGRRGCGRG fslp26 CGGGCCCCCTTCCCCCGCCCTCGGGGCGGGG fshp06 CGGGCTTCCCCCCCCCGGTCCCTCGGGGG fslp02 CGGGCCCCCTTCCCCCGCCCTCCGGCGGG fshp30 CGGGCTTCCCCCCCCCGGGKRGGGMGGG fslp03 CGGGCCCCCTTCCCCCGYCCTCRGGGCGGG fshp31 CGGGCTTCCCCCCCCCGGGKRGGGGRGG fslp22 CGGGCCCCCTTCCCCCGCCTGYTCCGRGG fshp32 CGGGCTTCCCCCCCCCGRGGKRGGRRGGG fslp23 CGGGCCCCCTTCCCCCGCCTGYTCCGGGG fshp33 CGGGCTTCCCCCCCCCGRGGKRGGRGRGG fslp05 CGGGCCCCCTTCCCCCGYGTTCCRGGGGG fshp37 CGGGCTTCCCCCCCCCGGGGGGGGGGGG fslp07 CGGGCCCCCTTCCCCCGCGRTYCCRGGRGG fshp38 CGGGCTTCCCCCCCCCGCCCTCGGGGG fslp08 CGGGCCCCCTTCCCCCGGYCCCKGGGGG fshp08 CGGGCTTCCCCCCCCCGCCTCGRGRGGG fslp10 CGGGCCCCCTTCCCCCGCYGRTCCCRKGGR fshp09 CGGGCTTCCCCCCCCCGGGTCGCGTGTGGGG fslp24 CGGGCCCCCTTCCCCCGCCTGRYTCCGGRGG fshp10 CGGGCTTCCCCCCCCCGCCGGGGG fslp25 CGGGCCCCCTTCCCCCGCTGRYTCCGGGRGG fshp11 CGGGCTTCCCCCCCCCGGTCGCGSGTGGGG fslp12 CGGGCCCCCTTCCCCCGCTGTTCCGGGGG fshp13 CGGGCTTCCCCCCCCCGCCGGGGGGGGGG fslp13 CGGGCCCCCTTCCCCCGTGTYCCTCRGCGG fshp14 CGGGCTTCCCCCCCCCGCCGGGGGGG fslp27 CGGGCCCCCTTCCCCCGGTTCCTCGGCCGGG fshp15 CGGGCTTCCCCCCCCCGTTCCGGGGGG fslp29 CGGGCCCCCTTCCCCCGTCCTCGGGCGGG fshp34 CGGGCTTCCCCCCCCCGCTCCGGRMGGG fslp30 CGGGCCCCCTTCCCCCGTGGGTCCCGGGG fshp17 CGGGCTTCCCCCCCCCGCCGGGGGG fslp31 CGGGCCCCCTTCCCCCGCGGTCCRGMKGGG fshp35 CGGGCTTCCCCCCCCCGCCCGGRGGG fslp32 CGGGCCCCCTTCCCCCGCTGTCCCGGGG fshp19 CGGGCTTCCCCCCCCCGGTTCCGGGGG fslp33 CGGGCCCCCTTCCCCCGGGCCCGCCGGG fshp21 CGGGCTTCCCCCCCCCGGGGGGGGGGGGG fslp34 CGGGCCCCCTTCCCCCGTCCGCGTGCCGGG fshp22 CGGGCTTCCCCCCCCCGGGGGGGGGGG fslp35 CGGGCCCCCTTCCCCCGCGGWCCGCCGGG fshp23 CGGGCTTCCCCCCCCCGGGGGGCC fslp36 CGGGCCCCCTTCCCCCGGGBCCMGCCGRGG fshp26 CGGGCTTCCCCCCCCCGCCCGGRGGGGGG fslp37 CGGGCCCCCTTCCCCCGGRRCCCMRCCGGG fshp39 CGGGCTTCCCCCCCCCGTTCCGGCGGG fslp38 CGGGCCCCCTTCCCCCGCGTCCCCGGG fshp40 CGGGCTTCCCCCCCCCGTCCCGGGGGG fslp39 CGGGCCCCCTTCCCCCGRWCCGCCCGGG fshp41 CGGGCTTCCCCCCCCCGCCCRGGGGGGGG fslp40 CGGGCCCCCTTCCCCCGGGCCRCCMGGG fshp42 CGGGCTTCCCCCCCCCGGCGCGGCGGGG fslp41 CGGGCCCCCTTCCCCCGGCCGCGGGG fshp43 CGGGCTTCCCCCCCCCGCTGCGCGGG fslp42 CGGGCCCCCTTCCCCCGCCGCCGCGGG fshp44 CGGGCTTCCCCCCCCCGTTCCCCGGGGG fslp43 CGGGCCCCCTTCCCCCGCGGTCCGCTGGGG fshp45 CGGGCTTCCCCCCCCCGCCCCGGGGG fslp44 CGGGCCCCCTTCCCCCGCCCCRRGRCRGG fshp46 CGGGCTTCCCCCCCCCGGGRGGGGRGGGG fslp45 CGGGCCCCCTTCCCCCGCCCRRGRCRGGG fshp47 CGGGCTTCCCCCCCCCGCCCCGCCCGGGG fslp46 CGGGCCCCCTTCCCCCGTCCCCGCGGG fshp48 CGGGCTTCCCCCCCCCGGCCCCGGGGGG fshp49 fshp50 fshp51 fshp52 fshp53 fshp54 fshp55 CGGGCTTCCCCCCCCCGGTGCMTCGRGRGGGG CGGGCTTCCCCCCCCCGGTCCCCGGGGG CGGGCTTCCCCCCCCCGGGGCGGG CGGGCTTCCCCCCCCCGMKCGGTGCCGGG CGGGCTTCCCCCCCCCGGGCGRTGCCGGG CGGGCTTCCCCCCCCCGGGTGCGRCCGGG CGGGCTTCCCCCCCCCGTTCCGGGGGG Pre-amplification primers (H5 and H7 specific): prfh5eh RYTTYTTGCTCCGWTGCTC prrh5eh TCCCCGTCTCCTKCCYTG = H5-kha-3: TCCCCGTCTCCTKCCYTG prfh5wh TTTTGCTCCYGTTGCRTC prrh5wh TCCYCCRTCHCCTTCCTT prfh7eh TGYCCGGTTGTWRC lt: GK7.4: TTTGTTCTGCGCGTTC prrh7eh TTTGTTCTGCHGCGTYC prfh7wh.1 GCCCTCGRTTGTCRC Detection primers: prfh7wh.2 CCCTCGRTTGTCRGC fdhp Fam-isoC-CGGGCTTCCCCCCCCCG prrh7wh TTTGTRTCGCTGCGTYC fdlp CL orange 560-isoC-CGGGCCCCCTTCCCCCG 484 PRIMER SYSTEM COVERING LL IV CLEVGE SITES TO DTE
N e w m e t h o d f o r I V ( N D V ) p a t h o t y p i n g HP Preamplification Multiplex PCR LP
E a s t e r n h e m i s p h e r e r e s u l t s
W e s t e r n h e m i s p h e r e r e s u l t s
R e s u l t s f o r c l i n i c a l s p e c i m e n s
P u b l i k a t i o n e r
Normalized F(CL Orange 560)F(FM) N D V p a t h o t y p i n g 0.5 0.4 0.3 0.2 0.1 0-0.1-0.2 HK 99/75 HK 138/80 Queensland V4/66 Ulster/67 SE/94 BG 13/69 C 12/63 Eawood/62 LaSota Beaudette C B1 NTC -0.3-0.4 0 5 10 15 20 25 30 Genotype I lentogenic strains (dashed lines), Genotype II meso/velogenic strains (solid lines) and lentogenic strains (dotted lines) Cycle Number
T h e S m a r t C a p S y s t e m Matrix gene amplification (CRL) (Taqman detection Cy5) H5/H7 amplification (CRL) (No detection) The Cepheid SmartCycler Pathotyping two-level reaction
N D V p a t h o t y p i n g o n p o r t a b l e P C R i n s t r u m e n t
C O N C L U S I O N S This method has the potential to substitute CS sequencing for determination of the molecular pathotype. Rapidly providing crucial information concerning NI outbreaks which may be critical to their management. The new assay may be extended to the LP/HP differentiation of previously unknown H5/H7 isolates. It may be considered for integration into surveillance and control programmes both in domestic and wild bird populations.