[Application of fluorescent real-time polymerase chain reaction in analyzing the epidemic of influenza among children in Guangzhou area in 2006].

OBJECTIVE: To investigate the prevalence of influenza virus infections in children in 2006 using the real-time PCR method. METHODS: (1) Consulting the most conserved sequence NP gene of influenza virus, after comparing with the NP gene sequences of influenza virus in GenBank, one pair of specific primers and one TaqMan probe were designed for each subtype of influenza virus by the software Primer Express. The sensitivity of influenza was evaluated by testing known positive samples which had been two-fold diluted. The specificity of real-time PCR for influenza virus detection was assessed by cross testing 60 isolates of influenza A, 16 isolates of influenza B, and by testing a variety of other respiratory viruses positive samples; (2) 281 nasopharyngeal aspirate samples were detected by real-time PCR and virus isolation; (3) the 12 301 specimens from the patients of Guangzhou Children's Hospital were tested by using the real-time PCR method. Furthermore, the real-time PCR reagent was evaluated by comparing with the result of virus isolation. RESULTS: (1) The sensitivity of real-time PCR developed in this study for influenza A detection was 1:2(22) and for influenza B was 1:2(20) in two-fold serially diluted way. (2) No positive results were found in cross testing of other viruses positive specimens. (3) Influenza virus was detected from 1687 cases (13.71%) out of the 12 301 cases, including 773 cases (45.8%) positive for subtype A and 914 cases (54.2%) positive for subtype B; 455 out of 525 (86.7%) of influenza B positive specimens and 70 out of 525 (13.3%) of influenza A (H1N1) positive specimens were from patients seen during January to April; 419 out of 1118 (37.5%) specimens positive for influenza B and 699 out of 1118 (62.5%) specimens positive for influenza A (H1N1) were from patients seen from May to August. Influenza virus could be identified from 1380 samples by the methods of virus isolation, accounting for 81.80% of the 1687 positive samples detected by real-time PCR. All the influenza virus subtype A was H1N1. CONCLUSION: The real-time PCR method developed in this study was sensitive and specific for detecting influenza A and B in clinical specimens. During 2006, influenza A and influenza B co-circulated. The predominant virus was influenza B from January to April, peaking in April. Influenza A (H1N1) prevailed from May to August, with the peak in June.

[Sequence analysis of VP7 gene from rotavirus field strain from Guangzhou, China].

OBJECTIVE: To compare the difference of VP7 fragment at the nucleotide level between rotavirus Guangzhou field strain R97-196 and rotavirus Beijing field strain or prototype strains. METHODS: The VP7 fragment amplified from Guangzhou field strain R97-196 by RT-PCR was cloned into the T-A cloning vector pUCm-T and sequenced. RESULTS: The VP7 fragment from Guangzhou field strain R97-196 was 1,062 bp in length and contained two open reading frames which is consistent with that reported in the literature. The sequence analysis revealed that the cDNA from R97-196 shared higher nucleotide and amino acid identities with Beijing field strain T73 (98% and %, respectively) than with serotype 2-4 (G types) rotavirus (from 74% to 77% and 73% to 81%, respectively). The divergence of amino acid sequences is mainly within the nine divergence regions. The phylogenetic analysis revealed that the VP7 fragment from R97-196 was far away from rotavirus serotype G1 strain Wa. CONCLUSIONS: The VP7 gene fragment from rotavirus Guangzhou field strain R97-196 belongs to rotavirus serotype G1. And variation of rotavirus VP7 gene fragment seems to be a geographic matter.