CAPÍTULO 3: ESTUDIO DE FACTORES QUE INCIDEN EN LA CONSTRUCCION DE
3.2. Categoría 2 – Pluralismo, Transcendencia y libertad
Samples for peR
EHV-2 and EHV-5 peR: With some exceptions, PCR reactions using primers specific
for EHV -2 and EHV -5 were routinely performed on all the samples that showed herpesviral CPE in cell culture. On most occasions, where a CPE was observed on
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EHV- 114 5,'erology 73
R K- 1 3 cells it was also present in the EFK cells. In such cases, occasionally, if PCR specific for either EHV -2 or EHV -5 performed on lysate from one cell culture was positive, the corresponding cell culture w as not checked for the presence of the same virus. All the cell cultures that were positive for EHV -5 were also checked for the presence of EHV -2 DNA. Also, on a few occasions, PCR was performed on cell l ysates from cultures that did not show CPE. This happened mostly, but not exclusively, when CPE was present in a different cell l ine inocul ated with the same sample.
EHV- J and EHV-4 peR: The following samples were checked for the presence of
EHV- 1 and EHV-4 DNA: all PBL cultures on EFK cells, PBL cultures on RK- 1 3 cell s collected from routinely sampled foals a t the time when they showed serological evidence of recent EHV - 1 /4 infection, and all cell cultures positive for EHV -5. Additionall y, PCR with primers specific for EHV - 1 and EHV -4 were performed directly on all nasal swab filtrates.
peR reactions
The PCR primers and programs used i n this study are l isted in Table 3 . 1 . The reaction mix consisted of 0.2 mM of each dNTP, 1 IlM of each primer, and 1 .5 units of Taq DNA pol ymerase in I x Taq PCR reaction buffer ( 1 0 mM Tris-HCl, 1 .5 mM MgCb, 50
mM KCI , pH 8.3) in a 25
IJ.l
total volume. All PCR reagents were purchased fromRoche, and primers were custom synthesised b y Gibco, BRL. Reactions were performed i n thin-walled PCR tubes (Biological™ Continental Laboratory Products Inc. ) in a Perkin Elmer PCR 9600 thermocycler. For multiple reactions, a PCR master mix was prepared. PCR reagents were stored and used in a designated clean room, using dedicated pipettes, pipette tips and reagents. Preparation and addition of samples was performed in an area separated from the clean room and from the room where gel analysis of PCR products was performed.
Chapter 3 74
Table 3. 1 : peR primers and programs used in the study
PCR Primers Program ' Target Product Re!
size Forward: 5' -AGAAAATGGCACAGAGCCAG-3' -95 °C-5 min EHV-2 - 35 cycles: gB 257 bp Reverse: 5'-TGGCAATAAAATGGAGACTGC-3' 95°C - 60 s Forward : 60°C -90 s 5'-GAGACCACGTTGTCCCCG-3' 72 °C -60 s EHV-5 gB 251 bp Reverse: _4°C 5' -GCTTCAAGTCCCTCA TG AGC-3' Forward: 5' -GCGAGA TGTGGTTGCCT AA TCTCG-3' -94 °C-5 min EHV- I
EHV - 1 14 rever e2: gC 649 bp
-30 cycles: 5' -GAGACGGTAACGCTGGTACTGTTAA-3' 94°C - 75 s
EHV4 forward: 60°C -90
5' -ACGCACGAACAACTCAACCGATGT -3 ' 72 °C -90
EHV-4
EHV - 1 14 rever e2: _4°C gC 507 bp
5 ' -GAGACGGT AACGCTGGTACTGTT AA-3'
I B lack dots indicate steps in a PCR program
2 EHV- I and EHV-4 peci fic PCR reactions were performed as mul tiplex PCR reaction with all three pri mers used in one tube
3 Reubel et al. ( 1 995 ) 4 Lawrence et al. ( 1 994)
Preparation of samples for peR
3
4
DNA was not extracted for routine PCR screening. After freeze-thawing, aliquot of infected cell culture ly ate ( 1 00 � ) were tran ferred to Eppendorf tube and incubated at 95 QC for 20 minutes. Four � of this preparation was used in a 25-� PCR reaction.
On a few occasions, DNA was extracted from samples that were negative on PCR
despite producing CPE in cell culture. In these cases a DNA extraction kit ( QIAamp
blood kit, QIAGEN) was used according to the manufacturer' instructions. Positive
and negative controls were included with every run of the test. Positive controls consi sted of cell cultures infected with respective viruse and prepared in the same way as survey samples. Negative control included equine DNA ( non-infected EFK treated in an identical manner to the urvey samples) and cells infected with EHV-2 (for EHV-5 PCR), E HV-5 ( for EHV-2 PCR), EHV- I ( for EHV-4 PCR) and EHV-4 ( for EHV- l
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EHV- 114 Serology 75
PCR) . Thus, EHV- l and EHV-4 infected cultures served as both negative and positive controls in EHV - 1 /4 multiplex PCR.
Analysis of amplified DNA
Aliquots ( 1 0 !J,l) of PCR products were subjected to electrophoresis through a 1 .5 % ethidium bromide (EtBr) stained agarose gel (Gibco, BRL) in TBE buffer at 100V for 40 to 60 minutes, visual ised under UV light and photographed using polaroid black and
white photographic film (Polaroid 667) . A molecular size marker (fX 1 74 RF DNNHae
III fragments, Gibco B RL) was included for reference on every gel. The results were confirmed by dot blot and Southern hybridisation with digoxigenin (DIG) l abelled probes specific for individual herpesvi ruses. A sample was considered positive i f a product of the correct size was visible on a gel and its specificity was confirmed by Southern hybridisation. Samples that were positive on a dot blot, but negative on a gel were also considered positive, as the detection limit of Southern hybridisation with
DIG-labelled probes i s higher than the detection l imit of EtBr staining (Holtke et al.
1 995). The PCR test was considered valid if the positive and negative controls showed expected results.
Preparation of dot blots
After gel analysis, PCR reaction tubes were placed again in a thermal cycler, heated to 95 QC for 1 0 minutes, and quickly chilled on ice. One � of each PCR product was spotted on a pre-marked membrane (Hybond N+, Amersham) , and the membrane was allowed to dry. The DNA was fixed to the membrane by UV cross li nking, by placing the membrane wrapped in a S aran Wrap, DNA side down, on an UV transillumi nator for 4 minutes. The blots were stored dry at 4 QC until use.
Preparation of probes
Virus speci fic probes were prepared b y random primed labelling of the ampli fication products from PCR reactions performed as described above with the reference viruses used as target DNA. The fol lowing reference viruses were u sed: EHV -2 86, EHV -5 2-
1 4 1 , EHV - 1 Durham, and EHV -4 Homer. The identities of amplified products were confirmed by sequencing before they were l abelled and used as probes. For l abelling, PCR products were gel purified after electrophoresis through a 1 .5 % EtBr stained low melting point agarose gel (Seaplaque) in T AE buffer at 1 00V for 60 minutes. Gel slices
Chapter 3 76
containing PCR products were melted at 70 °C in the presence of 4 � of GELase buffer per 200 mg of a gel slice (GELase™, Epicentre Technologies). After equilibration, 1 unit of GELase enzyme per 200 mg of gel was added and the mixture incubated at 45 °c for 45 minutes. Finally, the DNA was ethanol precipitated with 5 M ammonium acetate, the pel let washed with 70% ethanol, dried under vaccum for 5 minutes (Savant Speedvac SC I OO) and resuspended in 10 � dH20. A small aliquot of this preparation was run on a 1 .5 % EtBt stained agarose gel in TBE buffer, and the amount of recovered DNA estimated by comparison of the intensity of the band with the DNA mass l adder included on the gel (Gibco, BRL). Approxi mately 1 80-360 ng of DNA was used in the subsequent l abelling reaction.
Labelling with DIG was performed using DIG-High Prime (DIG labelling and detection kit, Roche) according to the manufacturer' s instructions. Briefly, DNA templates diluted to 1 6 � with dH20 were heat denatured by boiling for 1 0 minutes and quickly c hi lling on ice, before 4 � of DIG-High Prime was added to every reaction, and the tubes incubated at 37 °C overni ght. Reactions were stopped by adding 2 � of 200 mM EDTA, pH 8.0. The yield of labelled DNA was estimated by spotting appropriate dilutions of the l abelled products on DIG quantification teststrips and comparing the intensity of dots obtained after immunological detection with the i ntensity of corresponding dots on the control strips according to the manufacturer' s instructions (Roche). For hybridisation, probes were used at a concentration of 2.5 ng/m! (EHV -5) or 5 ng/ml (EHV-2, EHV- l , and EHV-4).
Sequencing
Sequencing of the PCR products was performed using AmpJiC ycle ™ Sequencing Kit (Perkin Elmer) according to the manufacturer' s instructions. Four sequencing reactions, one with each of the four termination mixes, were performed for every template. The termination mixes consisted of 22.5 jlM 7-deaza-dGTP, 10 j.1M solution of each dATP, dCTP, dTTP, and 600 j.1M of one of the four ddNTPs (ddATP, ddTTP, ddCTP, ddGTP). The sequencing reactions consisted of 0.5 jlM of primer, 2 jlCi [a-33P] -dCTP, 50 mM Tris-HCl, pH 8.9, 10 mM KCI , 2.5 mM MgCh, 0.025 % v/v Tween® 20, 1 unit of AmpliTaq® DNA polymerase (cycle sequencing), 40 ng of template DNA, 0.5 j.1M of additional dA TP/dTTP and 2 � of one of the four termination mixes. The cycling
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EHV- J/4 Serology 77
temperatures were as follows: denaturation at 95 °C (60 seconds) fol lowed by 25 cycles of denaturation at 95 °C (30 seconds), annealing at 60 °C (30 seconds) and elongation at
72 °C (60 seconds). At the end of the nm, the samples were cooled to 4 °C, removed
from the thermal cycler and 4 � of stop solution (95% Formamide, 0.05% B romophenol Blue, 0.02% Xylene Cyanole FF, 20 mM EDT A) was added to every tube. The reactions were either analysed immediately or stored at -20 °C for up to one week. A 6% polyacrylamide/urea sequencing gel was prepared according to the
standard protocol (Sambrook et al. 1 989). The amplification products were denatured
by boil ing for 5 minutes and quickly chilling on ice. Then, 3 � of the denatured DNA from each of the four termination reactions (in the order: G, A, C, T) of one template was loaded into the wells of the gel. The well s were thoroughly rinsed i mmediately prior to loading and the samples subjected to electrophoresis at 70 W for 2 85 minutes. After the first 240 m inutes, the electrophoresis was stopped and 3 �l of each of the termination reaction mixes loaded again into the additional wells, before sequencing products were subjected to electrophoresis for an additional 95 minutes. Thus, long and short runs were performed for every sample. Fol lowing electrophoresis, the gel w as disassembled from the gel sandwich, fixed with gel fixing solution (5% methanol, 5 % glacial acetic acid in dH20) for 1 5 minutes, transferred t o 3 M M Whatman fil ter paper and dried for 40 minutes in a gel dryer (BioRad, model 583). The dried gel was exposed to an X-ray film for 1 2-24 hours at RT, developed, and the sequence read manually from the autoradiogram.
Hybridisation with specific probes
The following hybridisation temperatures were used: EHV-2
EHV-5 EHV- l EHV-4
45 °C
The membrane was pre-hybridised in 20 ml of hybridisation buffer (DIG Easy H yb, Roche) per 1 00 cm' of the membrane, at the appropriate hybridisation temperature, for at least 1 hour. Directly before use, the relevant probe was heat denatured by boiling for 1 0 minutes in a water bath and quickly chilling on ice. The denatured probe was then diluted in DIG Easy Hyb to the desired concentration (see "probe preparation") and
Chapter 3 78
added to the hybridisation bag, from which the pre-hybrid isation solution had been removed. The hybridisation was performed overnight. After hybridisation, the membrane was washed twice in 2 x wash solution (2 x SSC, containing 0. 1 % SDS) for 5 minutes at RT, and then twice i n 0.5 x wash solution (0.5 x SSC, containing 0. 1 % SDS) for 1 5 minutes at 68 QC in order to remove u nbound probe. The hybridisation solution containing the probe was saved for re-use. Re-used probes were stored at -20
QC and denatured at 68 QC for t o minutes prior to use in any subsequent hybridisations.
Detection of DIG labelled probes
DIG-labelled probes were detected colorimetrical l y using a DIG labelling and detection kit according to the manufacturer's instruction (Roche) . The reagents llsed in the detection procedure are l isted in Appendix E. All incubation steps were performed at RT with agi tation. Amounts of solutions given are for detection of a 1 00-cm3 blot.
After hybridisation and hybridisation washes, the membrane was equilibrated in washing buffer for 1 minute. The membrane was then incubated in a series of solutions as follows:
• 1 00 ml of blocking solution for 30-60 minutes
• 30 ml of alkaline phosphatase conj ugated anti-DIG antibody, diluted in blocking
solution to a concentration of 1 50 mU/ml for 30 minutes
• 1 00 ml washing buffer for 1 5 minutes, twice
• 20 ml detection buffer for 2-5 minutes
After equilibration in the detection buffer, the membrane was sealed in a plastic bag with 1 0 ml color substrate solution, and incubated in the dark, without agitation, until the desired intensity of spots was reached (usually 3 to 6 hours) . The reaction was stopped by washing the membrane in dH20, and results documented by photocopying the wet fil ter. Usually, two to three membranes were developed at the same time.
Re-probing
Color precipitate was removed by incubating the membrane at 55-60 QC in dimethylformamide (Sigma) for several hours. After the blue colour was no longer visible, the membrane was washed in dH20, and incubated twice, 1 0 minutes per i ncubation, in alkaline-probe stripping solution (0.2 M NaOH, 0. 1 % SDS) at 37 QC. The
EHV-l/4 Serology 79
membrane was then washed in 2 x sse, and pre-hybridised before hybridising with the
next probe.