Tamra

To measure the amount of phagocytosis, bacteria was labeled with the fluorescent dye TAMRA (Life Technologies, NY) by incubating MRSA suspended in PBS with TAMRA dye (12.5μg/ml) on ice for 10min in the dark followed by two washes with PBS to remove the excess dye. Adult and neonatal mice were intranasally inoculated with 2.0 x 10⁷ CFU’s of TAMRA-MRSA or unlabeled MRSA as a control. Whole lungs were removed at 30 min post-infection and a single cell suspension was prepared as described above. The cells were fixed with 4% paraformaldehyde and extracellular fluorescence was quenched using 0.4% trypan blue immediately prior to running on the flow cytometer. The percent of lung cells that expressed TAMRA labeled MRSA was measured using cells from mice exposed to unlabeled MRSA as the gating control.

Lima qPCR for genus amplification (Lima-genus) was based on a previously published protocol [7 (link)] modified from Gama et al [21 (link)] to improve sensitivity and reduce costs. Briefly, the reaction was prepared using 2.5 μL of genomic DNA, 12.5 μL of 2x TaqMan® Universal PCR Master Mix, 500 nM of each genus-specific primer and 300 nM of the probe labeled with FAM™ and TAMRA™ (Applied Biosystems, Foster City, CA, USA). The threshold cycle (Ct) cut-off value of 37.28 was set based on the ROC curve as described before [7 (link)].

Primer designing was performed with the Primer Premier v5.0 and Oligo v6.0, by applying the following criteria: PCR product size from 70 to 200 bp; Tm values from 57 to 64 °C and GC content from 30 to 60%. The obtained primer pairs were then checked for non-specific hybridizations in other genome regions with the NCBI Basic Local Alignment Search Tool (BLAST) at http://blast.ncbi.nlm.nih.gov/. The primer pair for each X-InDel locus was initially tested in a singleplex PCR reaction to evaluate the performance with Qiagen Multiplex PCR kit (Qiagen, Hilden, Germany). PCR amplification was carried out on GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA). Successful PCR products were then Sanger sequenced. After confirming the exact length and sequencing information of candidate InDels, all the markers were then schematically organized by expected amplicon length and assigned into three different dye-labelling fluorochromes (FAM, HEX, TAMRA) (Applied Biosystems, Foster City, CA).

CVB4E2 positive strand RNA was quantitated by two-step quantitative RT-PCR as described previously (25 (link)). Briefly, total RNA was extracted with RNeasy minikit (Qiagen, Valencia, Calif.) and resuspended. Total RNA was measured by a quantitative RT-QPCR for RNA with the Affinityscript QPCR cDNA synthesis kit and the brilliant II QPCR kit (Agilent technologie stratagene). Positive strand specific RT was carried out on extracted RNA by using the reverse primer at 42°C for 15 min. PCR was performed with universal cycle conditions (10 min at 95°C, 40 cycles of 30 s at 60°C) on a Mx3000p (stratagene). The following primers, used to detect CVB4E2 RNA, were located within the enterovirus 5′-nontranslated region, which is highly conserved among enterovirus serotypes: CVB4 forward (5′-CCC TGA ATG GGG CTA ATC) and CVB4 reverse (5′-ATT GTC ACC ATA AGC AGC CA). The sequence of the CVB4 probe was 5′-VIC-AAC CGA CTA CTT TGG GTG TCC GTG TTT-TAMRA (Applied Biosystems). The absence of contaminating DNA in samples was checked by RT-PCR without the reverse transcriptase enzyme. Primers and probe pairs were designed with PrimerExpress software, and the data were analyzed with Sequence Detector version 1.6.3 (both from Perkin-Elmer, Boston, Mass. Results were expressed as cycle threshold (Ct) which is inversely proportionate to RNA level.

Viral load in various tissues following MVA or VACV s.s. was determined by quantitative real-time PCR. Briefly, DNA was purified using the DNeasy Mini Kit (Qiagen, Valencia, CA). The primers and TagMan probe used in the quantitative PCR assay are specific for the ribonucleotide reductase Vvl4L of VACV. The sequences are: (forward) 5′-GAC ACT CTG GCA GCC GAA AT-3′, (reverse) 5′-CTG GCG GCT AGA ATG GCA TA-3′, (probe) 5′-AGC AGC CAC TTG TAC TAC ACA ACA TCC GGA-3′. The probe was 5′-labeled with FAM and 3′-labeled with TAMRA (Applied Biosystems, Foster City, CA). Real-time PCR was performed with the Bio-Rad iCycler iQTM Real-Time PCR Detection System (Bio-Rad Laboratories). Thermal cycling conditions were 50 °C for 2 min and 95 °C for 10 min for one cycle, followed by 45 cycles of amplification (94 °C for 15 s and 60 °C for 1 min). Standard curve was established from DNA of an MVA or VACV stock with previously determined titer. Corresponding CT values obtained by the real-time PCR reactions were plotted on the standard curve to calculate viral load in the samples. The number of viral DNA copies was normalized to that in the skin samples of uninfected naive mice.

Primer designing was performed with the Primer Premier v5.0 (PREMIER Biosoft, Palo Alto, CA, USA), applying the following criteria: PCR product size from 70 to 300 bp; Tm values from 52 to 60 °C and GC content from 30 to 60%. The “PIGtail” or partial tails of the original GTTTCTT nucleotide sequence were added to the 5’end of the unlabeled primers to promote full adenylation34 (link). The obtained primer pairs were examined for potential interactions with each other using AutoDimer software35 (link). They were also checked for non-specific hybridizations in other genome regions using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) at http://blast.ncbi.nlm.nih.gov/Blast.cgi. All the markers were then schematically organized by expected amplicon length and assigned into four different dye-labeling fluorochromes (FAM, HEX, TAMRA, and ROX) (Applied Biosystems, Foster City, CA).
Optimizations of the multiplex assay were performed on the basis of primer concentrations and peak heights. After that, 210 collected Chinese samples from Han and Tibetan individuals were genotyped with the developed PCR system. Based on the size information of each haplotype, Panel and Bin files were programmed.