Resuspended là gì


Hepatitis B virut (HBV) is a well-known agent of adễ thương & chronic hepatitis, with an estimated 350 million chronic carriersaround the world. HBV has a circular & partially doublestrandedDNA genome of 3.2 kb containing four overlapping open reading frames. HBV strains isolated worldwide have sầu been classified inkhổng lồ six genomic groups deduced from genome comparisons và designated genotypes A to lớn F (3, 6, 7). The HBV genotypes have sầu a characteristic geographic distribution.HBV genotyping by phylogenetic analysis based on nucleotide sequences produces the most reliable and certain genotyping results. However, this is not an appropriate method for largescale genotyping. On the other hvà, several groups have sầu reported the genotyping of HBV by the restriction fragment length polymorphism method (1, 2, 4, 8). However, their methods were not so sensitive & specific compared with our PCR genotyping method. In this paper, we report a simpler, more rapid, và more specific genotyping system for HBV involving PCR using type-specific primers.A total of 55 HBV DNA-positive sầu serum samples obtained from individuals in six different countries,including Japan, Vietphái mạnh, the United States, Egypt, Ghamãng cầu, & Bolivia, were used for the evaluation of our genotyping system. We selected the HBV DNA-positive sầu samples by nested PCR. The sequences of PCR primers used in this study are shown in Table1. The first-round PCR primers (outer primer pairs) and second- round PCR primers (inner primer pairs) were designed on the basis of the conserved nature of nucleotide sequences in regions of the pre-S1 through S genes, irrespective of the six HBV genotypes. P1 (sense) và S1-2 (antisense) were universal outer primers (1,063 bases). B2 was used as the inner primer (sense) with a combination called mix A for genotypes A, B, and C. Mix A consisted of antisense primers BA1R (type A specific), BB1R (type B specific), and BC1R (type C specific). B2R was used as the inner primer (antisense) with a combination called phối B for genotypes D, E, và F. Mix B consisted of sense primers BD1 (type D specific), BE1 (type Especific), & BF1 (type F specific). These primer combinations for second-round PCR were designed on the basis of the differences in the sizes of the genotype-specific bands. The type-specific primers were designed on the basis of the conserved nature of those sequences within a genotype và on the basis of their poor homology with the sequences derived from other HBV genotypes. The strategy for HBV genotyping isillustrated in Fig. The nucleic acid was extracted from 100-ml serum samples using a nucleic acid extraction kit (SepaGene RV-R; Sanko Junyaku Co., Ltd., Tokyo, Japan). The resulting pellet was resuspended in RNase-không lấy phí water & then subjected to lớn nested PCR. We amplified the HBV genome by nested PCR using the universal primers (P1 và S1-2) for the outer primers, followed by two different mixtures containing type-specific inner primers as described above sầu. The first PCR was carried out in a tube containing 40 ml of a reaction buffer made up of the following components: 50 ng of each outer primer, a 200 mM concentration of each of the four deoxynucleotides, 1 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, Conn.), và 13 PCR buffer containing 1.5 mM MgCl2. We used AmpliTaq Gold DNA polymerase to lớn obtain an automatic hot-start reaction.The thermocycler (GeneAmp PCR system 2400, 9600,& 9700; Perkin-Elmer) was programmed to first incubate the samples for 10 min at 95°C, followed by 40 cycles consisting of 94°C for 20 s, 55°C for đôi mươi s, và 72°C for 1 min. As illustrated in Fig. 1, two second-round PCRs were performed for each sample, with the common universal sense primer (B2) & mix A for types A through C & the comtháng universal antisense primer (B2R) và set B for types D through F.

A 1-ml aliquot of the first PCR hàng hóa was added to two tubes containing the second sets of each of the inner primer pairs, each of the deoxynucleotides, AmpliTaq Gold DNA polymerase, and PCR buffer, as in the first reaction.

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These were amplified for 40 cycles with the following parameters: preheating at 95°C for 10 min, trăng tròn cycles of amplification at 94°C for trăng tròn s, 58°C for đôi mươi s, and 72°C for 30 s, & an additional trăng tròn cycles of 94°C for đôi mươi s, 60°C for 20 s, & 72°C for 30 s. Genotypes of HBV for each sample were determined by identifying the genotype-specific DNA bands. The two different second-round PCR products from one sample were separately electrophoresed on a 3% agarose gel, stained with ethidium bromide, & evaluated under UV light. The sizes of PCR products were estimatedaccording lớn the migration pattern of a 50-bp DNA ladder (Pharmacia Biotech, Uppsala, Sweden). To thử nghiệm the validity of our PCR genotyping system, genotypes of HBV were also determined by phylogenetic analysis of pre-S1 through S genes in 40 samples. Amplified PCR products were subjected to lớn direct sequencing, and then phylogenetic analysis was performed as reported previously (5).Mix A allows for the specific detection of PCR products for types A, B, và C, & mix B allows for detection of types D,E, và F. As shown in Fig. 2, type-specific PCR products were recognized clearly by their distinct sizes in gel electrophoresis.When 28 isolates in the panel (5 samples from each genotypeexcept for type E), for which serum samples were available,were typed by PCR, the results were in complete accord with the sequences corresponding lớn their type-specific primers. In the second stage of PCR, type A HBV DNA was amplified with the type A-specific primer, but not with other type-specific primers. Furthermore, to confirm the specificity of our PCR assay, we compared the genotyping results between typing by PCR và by phylogenetic analysis for the 40 samples examined.The results showed 100% concordance between the two assays. In addition, the alignment of the representative pre-S1 through S genes of HBV isolates in the present study revealed that there was a consensus sequence at the same nucleotide positions among mỏi different isolates from each genotype. Using this new assay system, we investigated the geographic distribution of HBV genotypes in various countries. The data showed that the distribution of the HBV genotypes in this study population was in accord with the known geographic distribution of HBV genotypes (Table 2).The genotyping of HBV is important to clarify the route and pathogenesis of the virus. In particular, the examination of sequence diversity ahy vọng different isolates of the virut is important, because variants may differ in their patterns of serolô ghích reactivity, pathogenicity, virulence, & response to therapy.On the other hand, HBV has genetic variations which correspond lớn the geographic distribution, and it has been proposed that HBV can be classified into six major genotypes (3, 6, 7). In designing the genotype-specific PCR primers, it is well established that not only higher matching in the entire sequences but also the matching of the two to lớn three nucleotides at the 39 ends is one of the important parameters for specific priming. Based on this fact, we designed type-specific PCR primers. Sequences within the same genotype were different by >=2 nucleotides aao ước the entire sequences of the genotype-specific primer, while the sequence within the different genotypes had a difference of =of the results of PCR typing, phylogenetic analysis in the pre-S1 through S genes of HBV was also performed, and we confirmed the specificity of the results obtained with our PCR genotyping system. This method is very convenient & will assist research workers in conducting large-scale epidemiological studies. Additional investigations, using the serum samples from other geographic regions, are required for the further classification và characterization of HBV. In fact, Stuyver et al. (9) reported recently the identification of a novel genotype of HBV (designated genotype G). For this purpose, our genotypingsystem using the PCR method introduced here will be useful.In conclusion, we reported on a newly developed precise PCR genotyping system using type-specific primers, allowing the identification of types A through F. This assay system may be useful for rapid & sensitive genotyping of the HBV genome when either epidemiological, pathological, or transmission studies of this agent are carried out in large scale.We thank Tetsutaro Sata (National Institute of Infectious Diseases) for his continuous encouragement during this study. We also thank Chiaki Miyoshi (International Medical Center of Japan), Ko-ibỏ ra Ishikawaand Yutaka Takebe (National Institute of Infectious Diseases),Vo Xuan Ouang and Banh Vu Dien (Cho Ray Hospital, Ho Chi Minc City, Vietnam), Abdel Rahman El-Zayadi (Cairo Liver Center, Cairo,Egypt), and Alfred M. Prince (Thành Phố New York Blood Center, Thủ đô New York,N.Y.) for providing valuable serum samples.This study was supported in part by a Grant-in-Aid for Science Research of the Ministry of Education, Science và Culture of Japan, by the Ministry of Health and Welfare of Japan, và by an International Medical Cooperation Research Grant of Japan. mình tất cả dịch sơ qua test tuy vậy cơ mà giờ anh dại vượt bắt buộc thấy không nên be bét ah các bạn làm sao góp mình với

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