This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruse...This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.展开更多
A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans （AF2）, a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion （F...A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans （AF2）, a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion （Fe^2＋） or elemental sulfur （S^0） as sole energy source, but oxidizes So more effectively than Fe^2＋, which is different from ATCC23270 and AF3. The G＋C content of AF2 is 51.8% （molar fraction）, however, ATCC23270 and AF3 strains have G＋C content of 63.7% and 64.8% （molar fraction）, respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin （rus） and subunit III of aa3-type cytochrome oxidase （coxC） genes are not detected in AF2, but Fe^2＋ oxidase （iro） gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream ofpurA gene, while it is located at downstream ofpetC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain ofA. ferrooxidans, and that phenotypic differences among the strains ofA. ferrooxidans are closely correlated with their genetic polymorphisms.展开更多
基金The Basic Rasearch Project of Shenzhen(JC200903190778A)
文摘This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.
基金Project(200805032) supported by the Scientific Research Program of Marine Public Welfare Industry of China
文摘A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans （AF2）, a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion （Fe^2＋） or elemental sulfur （S^0） as sole energy source, but oxidizes So more effectively than Fe^2＋, which is different from ATCC23270 and AF3. The G＋C content of AF2 is 51.8% （molar fraction）, however, ATCC23270 and AF3 strains have G＋C content of 63.7% and 64.8% （molar fraction）, respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin （rus） and subunit III of aa3-type cytochrome oxidase （coxC） genes are not detected in AF2, but Fe^2＋ oxidase （iro） gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream ofpurA gene, while it is located at downstream ofpetC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain ofA. ferrooxidans, and that phenotypic differences among the strains ofA. ferrooxidans are closely correlated with their genetic polymorphisms.
