Patients and RNA extraction
All patient samples were provided by the department of gastroenterology, Royal Free Hospital, London, UK. Ileal lymphoid tissues from 91 affected children were examined (median age, 7 years; range, 3–14; 77 boys). Developmentally normal paediatric controls (n = 70; range, 0–17 years; 47 boys) included: 19 children with normal ileal biopsies, 13 children with mild non-specific chronic inflammatory changes, three children with ileal lymphonodular hyperplasia (LNH) investigated for abdominal pain, eight children with Crohn's disease, one child with ulcerative colitis, and 26 children who had undergone appendicectomy for abdominal pain including appendicitis.
MV positive control material included two cases of SSPE and MV infected Vero cells. Negative control material included uninfected Vero cells, and human tissues, control RNA extracted from Raji cells (Applied Biosystems, Foster City, California, USA) and normal peripheral blood mononuclear cells.
Total RNA was extracted from fresh frozen biopsies, peripheral blood mononuclear cells, and MV infected and uninfected Vero cell lines using the Ultraspec-11 RNA isolation system (Biotecx Laboratories, Houston, Texas, USA). Total RNA was extracted from formalin fixed, paraffin wax embedded tissues using the Purescript™ RNA isolation kit (Gentra Systems, Minneapolis, Minnesota, USA).
Solution phase RT-PCR
Polymerase chain reaction (PCR) primers and probes to conserved regions of the MV Nucleocapsid (N), Haemagglutinin (H), and Fusion (F) genes were designed using Primer Express Software Version 1.5 (ABI Prism; Applied Biosystems). The specificity of selected sequences was checked using the NCBI Blast program (www.ncbi.nml.nih.gov/blast
). Table 1 shows the MV primer and probe sequences, amplicon sizes, and GenBank accession numbers used for designing PCR primers and oligonucleotide probes. In some instances primer sets overlap with each other (for example, the sequence of amplicon N1 overlaps partially with the N2 PCR amplicon).
Measles virus primer and probe sequences
For in situ PCR, oligonucleotide probes were modified at the 5` end by the addition of a biotin moiety and for Southern blot analysis probes were labelled at the 3` end with digoxygenin. For TaqMan quantitative reverse transcription PCR (RT-PCR), probes were dual labelled with the fluorescent molecule FAM at the 5` end and the quencher TAMRA at the 3` end.
Purified MV RNA (HU2) was used as a positive control to optimise PCR assays. The following optimal reaction conditions were used for each 25 μl reaction: 0.4mM dNTPs, 0.4μM forward and reverse primers, 2.5mM magnesium acetate, 5 U rTth DNA polymerase, 0.01 U AmpErase, and 1× EZ buffer. The EZ buffer consisted of 50mM bicine, 125mM potassium acetate, 40% (wt/vol) glycerol (pH 8.2) (Applied Biosystems); 50 ng of extracted RNA was used for each reaction. The following RT-PCR thermal cycling conditions were used on a 9700 PCR thermocycler (Applied Biosystems): 50°C for two minutes, 58°C for 30 minutes, 95°C for five minutes, then 40 cycles of 94°C for 20 seconds, 59°C for 20 seconds, and 72°C for 20 seconds, followed by an extension step at 72°C for 10 minutes.
Southern blot analysis
To confirm reaction specificity, solution phase RT-PCR was performed, as described above, on four affected children positive for MV by TaqMan RT-PCR (see below). MV F and H gene amplicons from MV infected Vero cells, SSPE brain, and ileal lymphoid tissues from four affected children, together with a no template control, were examined by Southern blotting5
using sequence specific probes (table 1). MV specific oligonucleotide probes were labelled at the 3` end with digoxigenin using a DIG oligonucleotide 3` end labelling kit (Roche Molecular Biochemicals, Mannheim, Germany), hybridised to the Southern blot at 50°C in 5× saline sodium citrate (SSC) with 0.2% sodium dodecyl sulfate for one hour, washed once in 5× SSC at 50°C for 10 minutes, once in 2× SCC at room temperature for 10 minutes, and once in 0.2× SCC at room temperature for 10 minutes. Hybrids were detected using the DIG luminescent detection kit (Roche Molecular Biochemicals).
Real time quantitative RT-PCR based on the 5` nuclease assay was performed on an ABI 7700 Sequence detector (Applied Biosystems).6,7
Sequence specific PCR primers and TaqMan probes were designed using Primer Express software as described above. All quantitative PCRs were prepared in a dedicated facility in a class 2 laminar flow bench hood using dedicated pipettors and aerosol resistant pipette tips. Template RNA was prepared and added to the PCR mastermix in a separate facility.
TaqMan RT-PCR was performed using EZ TaqMan RT PCR reagents according to the manufacturer's instructions (Applied Biosystems). RT-PCR reactions were performed in duplicate under the following conditions for each 25 μl reaction: 1× EZ buffer, 3mM MnOAc2, 200 nmol of each primer, 100 nmol of TaqMan probe, 0.01 U of AmpErase, 0.1 U of rTth polymerase, and 3 μl (5–50 ng) of total RNA. The thermal cycling conditions on the 7700 were as follows: 50°C for two minutes, 58°C for 30 minutes, 95°C for five minutes, followed by 40 cycles of 94°C for 20 seconds and 60°C for one minute.
Controls for TaqMan RT-PCR included the following: no template control (water added as template), no amplification control (omission of rTth polymerase), irrelevant target primers and specific TaqMan probe (human papillomavirus 16, human herpes virus 8 primers), probe only control (omit PCR primers), human RNA control, spiked RNA control, and asymmetric TaqMan PCR (TaqMan PCR with one or other primer and specific TaqMan probe).
A gene dosage correction was carried out using glyceraldehyde phosphate dehydrogenase as a housekeeping gene. Measles virus quantitative TaqMan RT-PCR was performed by generating standard curves for the F and H genes. Taqman RT-PCR standards were generated by cloning the F and H gene specific PCR products into a vector using the TOPO TA cloning™ system (Invitrogen, Groningen, the Netherlands), according to the manufacturer's instructions. Plasmids containing the PCR inserts were then in vitro transcribed into cRNA using the Riboprobe™ in vitro transcription system (Promega, Madison, Wisconsin, USA). Serial dilutions of this cRNA were used to generate standard curves.
RT in situ PCR
RT in situ PCR facilitates low copy gene detection and permits cellular localisation within tissues, with a reported sensitivity of one viral genome copy/cell.8–10
Sections were dewaxed in xylene and taken through a series of graded alcohols. Endogenous avidin and biotin activity was blocked using the Dako biotin blocking system (Dako, Glostrup, Denmark). Sections were digested with proteinase K (300 μg/ml) for 17 minutes at 37°C.
After pretreatment, MV RNA was amplified using the following protocol: 58°C for 45 minutes and 94°C for five minutes, followed by 25 cycles of 94°C for 45 seconds, 60°C for 45 seconds, and 72°C for 45 seconds. After amplification, sections were fixed in 100% ethanol and air dried.
Hybridisation was carried out with a 5` biotinylated oligonucleotide probe using previously published protocols.8
Hybrid detection was achieved using a three step immunocytochemical method11
or dinitrophenol tyramide signal amplification.12
Alkaline phosphatase was detected with nitrobluetetrazolium (NBT) and bromochloroindoylphosphate (BCIP) as chromogen. Endogenous alkaline phosphatase was blocked using levamisole, an endogenous AP inhibitor (Dako), during chromogenic detection.
Reaction optimisation experiments were initially carried out using formalin fixed, paraffin wax embedded, measles infected Vero cells, and a variety of probe concentrations (1 μg/ml, 1.5 μg/ml, 2 μg/ml). A concentration of 1 μg/ml yielded optimal signals and was used in subsequent experiments. Biopsies from 73 affected children and five normal controls were examined.
Controls for RT in situ PCR included the following: MV infected and uninfected Vero cells, a mixed population of infected and uninfected Vero cells, MV N gene primers and an irrelevant probe (nonsense pyruvate dehyrogenase probe), and irrelevant primers and N gene specific probe. Hybridisation control experiments were performed using a histone mRNA probe. Other control experiments included RNase digestion of MV infected Vero cells before RT in situ PCR.
Combined RT in situ PCR and immunohistochemistry
To examine MV signal localisation, RT in situ PCR for the MV N gene was performed as described above on tissue sections following immunohistochemistry using the follicular dendritic cell CNA 42 monoclonal antibody13
(Dako). For in situ hybridisation a 5` biotin labelled oligonucleotide probe was used as above.
The MV hybridisation signal was developed with horseradish peroxidase and aminoethyl carbazole (AEC; Vector Laboratories, Burlingame, California, USA), and the dendritic cell signal was developed using a three step detection method with AP (Dako) and NBT and BCIP (Roche Molecular Biochemicals) as substrate.
Approval for these studies was obtained from the ethical practices committee of the Royal Free, Hampstead, NHS Trust. Fully informed, written parental consent was obtained from all trust patients including controls.