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[27]. HCoV-NL63 RNA was detected using primers described by Mo?s et al. [28], and Boca DNA was detected using primers described by Allander et al. [23]. The primers used to detect KIV and WUV DNA were previously described by Allander et al. and Bialasiewicz et al. [25, 26]. 2.3.3. PCR and RT-PCR Conditions The RT step was performed for 60?min at 37��C in a 10?��L reaction volume containing 1X GeneAmp RNA PCR buffer, 5?��L of 25?mM MgCl2, 1?mM of each deoxynucleoside triphosphate, 2.5?��M of random hexamers, 0.5?��L RNase inhibitor, 3?��L of viral nucleic acid, and 2.5?U/��L reverse transcriptase enzyme (GeneAmp RNA Core Kit; Applied Biosystems, Chicago, IL). Following heat inactivation of CDK9 the reverse transcriptase at 90��C for 5?min, the entire reaction mixture was used for PCR in a total volume of 50?��L. The reaction mixture composition was 2?mM MgCl2 solution, 1X PCR buffer containing 0.02?pg of each forward and reverse primer, and 0.05?��L of 5?U/��L Ampli Taq DNA Polymerase (Thermo Fisher Scientific, Pittsburgh, USA). PCR was performed as follows: 94��C denaturation for 1?min, followed by 40 cycles of denaturation at 94��C for 30?sec, annealing at 50��C for 30?sec and elongation at 72��C for 30?sec, and a final extension at 72��C for 7?min. Water was used instead of nucleic acids as a negative control. The specificity of the PCR was established for each PCR format using Dolutegravir a panel of ATCC reference viruses to check for cross-reactivity to old respiratory viruses. DNA templates (110�C140?bp, Thermo Scientific) encompassing the annealing sites of the primers and probes were used as positive controls for the detection of nucleic acid from HCoV-NL63, hMPV, Boca, WUV, and KIV. 2.4. Statistical Analysis Data analysis was performed using the Statistical Package for the Social Sciences (SPSS version 20.0, IBM Corp, Armonk, NY, USA). The descriptive statistics of the continuous variables were compared using a nonparametric Mann-Whitney U test or Kruskal-Wallis test. For the categorical variables, a Chi-square or Fisher's exact test or Z-test was applied to test the difference between proportions or to assess whether any association existed between the proportions. The two-tailed probability value P selleck products significant. 3. Results From the overall number of 850 hospitalized patients three hundred fifty one patients (47.8%) were diagnosed with viral respiratory infections, 210 (59.8%) of them were males and 141 (40.2%) were females. Results show that from the 351 patients 408 viruses were detected. Table 1 shows that HRV was the most detected virus in clinical respiratory specimens of patients with respiratory symptoms (41.6%), followed by FluA (15.1%), RSV (13.1%), and HCoV-OC43 (12.3%). Among the 351 hospitalized patients viral mixed detection was detected in 49 patients (14%). HRV was the most common virus associated with mixed detection (7.1%), followed by AdV (4%), HCoV-OC43 (3.7%), RSV (3.1%), and FluA (2.8%) (Table 1).