Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method

Similar to other segmented RNA viruses, influenza viruses can exchange genome segments and form a wide variety of reassortant strains upon coreplication within a host cell. Therefore, the mapping of genome segments of influenza viruses is essential for understanding their phenotypes. In this work, we have developed an oligonucleotide microarray hybridization method for simultaneous genotyping of all genomic segments of two highly homologous strains of influenza B virus. A few strain-specific oligonucleotide probes matching each of the eight segments of the viral genomes of the B/Beijing/184/93 and B/Shangdong/7/97 strains were hybridized with PCR-amplified fluorescently labeled single-stranded DNA. Even though there were a few mismatches among the genomes of the studied virus strains, microarray hybridization showed highly significant and reproducible discrimination ability and allowed us to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates. Additionally, we were able to detect the presence of at least 5% of mixed genotypes in virus stocks even when conventional sequencing methods failed, for example, for the NS segment. Thus, the proposed microarray method can be used for (i) rapid and reliable genome mapping of highly homologous influenza B viruses and (ii) extensive monitoring of influenza B virus reassortants and the mixed genotypes. The array can be expanded by adding new oligoprobes and using more quantitative assays to determine the origin of individual genomic segments in series of reassortant strains prepared as vaccine candidates or in mixed virus populations.     

Autozygosity Mapping with Exome Sequence Data

Autozygosity mapping is a powerful method for the identification of recessively inherited disease genes using small inbred families. Typically, microarray SNP genotype data are first used to identify autozygous regions as extended runs of homozygous genotypes. Next, candidate disease loci are found by defining regions that are autozygous in all affected patients. Finally, the disease gene is identified by sequencing the genes within the candidate disease loci. However, with the advent of massively parallel sequencing, it is now possible to sample or to completely sequence an individual's genome, or, more commonly, exome. This opens up the possibility of concurrently defining autozygous regions and identifying possibly deleterious sequence variants, using data from a single sequencing experiment. Consequently, we have developed a set of computer programs that identify autozygous regions using exome sequence data. These programs derive their genotyping data either by the ab initio detection of all sequence variants or by the assessment of 0.53 million known polymorphic positions within each exome dataset. Using genotype data derived solely from exome sequence data, it was possible to identify the majority of autozygous regions found by microarray SNP genotype data.     

Measles virus genotyping by nucleotide-specific multiplex PCR

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.     

Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.     

Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis

The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.     

Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation

We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.     

A real-time Taqman method for hepatitis C virus genotyping.

BACKGROUND: There is the need for a rapid, inexpensive method for genotyping hepatitis C virus (HCV) to support clinical practice. OBJECTIVES: To develop a real-time (Rotor-Gene 3000) Taqman assay for HCV genotyping in a single tube. STUDY DESIGN: Seven type-specific probes, two for genotypes 1-3 and one for genotype 4 were designed around genotype-specific motifs in the 5' non-coding (NC) region to create two panels of probes. The first panel included two probes for genotype 1 detection and a single probe each for genotypes 2 and 3. The second panel had two probes for confirmation of genotypes 2 and 3 and a first line probe for genotype 4 detection. A comparative analysis of the Taqman assay against our in-house sequence-based method using 154 consecutive clinical samples, from HCV carriers in Cambridge, and four samples from the Quality Control for Molecular Diagnostics (QCMD) System was undertaken. RESULTS: 158 samples were analysed by conventional sequencing: 49% (n=78) were genotype 1, 11% (n=18) genotype 2, 30% (n=47) genotype 3 and 6% (n=10) genotype 4. For two samples, the sequence data was heterogeneous and difficult to analyse, suggesting mixed infection and for three samples, the viral load was insufficient for sequencing. Concordant results were obtained with the novel Taqman assay for 77/78 (99%) of genotype 1 isolates (positive with both genotype 1 probes), 17/18 (94%) of genotype 2 isolates, 43/47 (91%) of genotype 3 isolates and 10/10 (100%) genotype 4 isolates. One isolate, untypeable with sequencing was genotyped with the Taqman assay. CONCLUSIONS: The Taqman assay was sensitive, specific and reliable over a wide range of viral loads and could identify mixed infections. These results highlight the potential of the Taqman assay as a fast, accurate and convenient method for routine HCV genotyping.     

Molecular epidemiology of measles virus in Taiwan in 2010–2011: The common genotype changed from H1 to D9 and the first appearance of D4

Measles has been controlled effectively in some countries because of high coverage rates with an effective vaccine. However, measles outbreaks still occasionally occur in areas with high vaccine coverage as a result of imported transmission. To identify the sources of measles infection and to determine whether measles cases are part of a single outbreak or due to multiple importations, measles virus (MV) genotyping is required and plays an important role in MV elimination. In Taiwan, genotype H1 of MV was detected most frequently before 2009. From 2006 to 2011, 47 of 48 genotype H1 cases were associated with the imported cases, indicating that genotype H1 was not an endemic genotype in Taiwan after 2006. The distribution of the other genotypes (D3, D4, D5, D8, D9, and G3) detected during 2006–2011 varied by year. Taiwan has a pattern of measles genotypes that is consistent with the elimination of MV and with the absence of endemic genotypes. In this study, the genotypes of 40 cases of MV detected during 2010–2011 were investigated and analyzed. In 2010, the most common genotype changed from H1 (3/40) to D9 (35/40). In 2011, genotype H1 was not detected, and genotype D4 first appeared and was imported from Europe. The dynamic change of detected genotypes of MV in Taiwan is influenced by the activity of a measles control program in WHO regions. This study emphasizes that global synchronous elimination is important for an individual country or area to maintain free from MV. J. Med. Virol. 85: 1095–1099, 2013. © 2013 Wiley Periodicals, Inc.     

Clinical evaluation of two methods for genotyping hepatitis C virus based on analysis of the 5' noncoding region

We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5'NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5' noncoding (5'NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5'NC genotyping results. The sensitivities of the methods were assessed by using the 5'NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5'NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5'NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5'NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5'NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5'NC genotyping methods were comparable and dependent on the amplification test used ( approximately 10(3) IU/ml with the qualitative HCV RNA tests and approximately 10(5) IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5'NC test. In conclusion, the performance characteristics of the 5'NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.     

核酸试纸条法检测HBV基因型方法的建立

目的 建立一种快速、肉眼可观测的方法检测乙型肝炎病毒(HBV)基因型.方法 根据GenBank中已发表的明确HBV分型全序列,设计特异的引物和标记生物素探针,建立核酸试纸条法检测150例乙型患者和20例健康体检者的乙型肝炎基因型,同时采用荧光定量PCR法进行比较.结果 150例标本中B型检出率为34.00%(51/150),C型占61.33%(92/150),B/C混合型占4.00%(6/150),未分型占0.67%(1/150).与荧光定量PCR法进行比较,结果一致.结论 核酸试纸条法检测HBV基因型与荧光定量pCR法相比较灵敏度和特异性相似,但该方法更简便、快速,更适合临床开展.     

Development of a DNA microarray for simultaneous detection and genotyping of lyssaviruses

The lyssavirus genus of the Rhabdoviridae family of viruses includes 7 genotypes and several non-assigned isolates. The source of lyssavirus infections is diverse with numerous reservoirs in a wide geographical area. In many parts of the world reservoir hosts can potentially be carrying one of several lyssavirus strains and possibly new divergent isolates await discovery. Accordingly, generic detection methods are required to be able to detect and discriminate all lyssaviruses and identify new divergent isolates. Here we have allied a sequence-independent amplification method to microarray to enable simultaneous detection and identification of all lyssavirus genotypes. To do so, lyssavirus RNA was converted to cDNA and amplified in a random PCR, labelled and hybridized to probes on the microarray chip before being statistically analysed. The probes were to a 405 bp region of the relatively conserved N gene. Here we demonstrate a microarray capable of detecting each of the seven lyssavirus genotypes. The random amplification of lyssavirus RNA and the numerous oligonucleotide probes on the microarray chip also offer the potential to detect novel lyssaviruses.     

Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology

Staphylococcus aureus is a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed an in silico typing scheme for the software SeqSphere⁺ (Ridom GmbH, Münster, Germany). The implemented target genes (n = 182) correspond to those queried by the Identibac S. aureus Genotyping DNA microarray (Alere Technologies, Jena, Germany). The in silico scheme was evaluated by comparing the typing results of microarray and of WGS for 154 human S. aureus isolates. A total of 96.8% (n = 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosome mec element types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.     

DNA芯片检测乙型肝炎病毒基因多态性

目的　建立DNA芯片检测乙型肝炎病毒 (hepatitisBvirus,HBV)基因多态性的研究方法并对实验条件进行优化。方法　设计多条寡核苷酸探针 ,在硅烷化芯片的特定位置上 ,用点样仪将探针固定 ,并与PCR扩增的HBV基因相应区段杂交 ,杂交结果影印至硝酸纤维素膜 ,经BCIP NBT避光显色 ,用放大镜观察杂交信号呈暗紫色圆点 ,根据特定位置上杂交信号的有无和与之相应的探针序列来判定基因突变的类型。结果　通过 1次杂交反应可检测HBV前C C区 (nt 1896 1814 )、BCP区 (nt1762 1764)和P区 (nt 52 8 552 )等多个位点的变异 ,与测序分析结果完全一致 ,具有较好的检测灵敏度和重复性。结论　DNA芯片检测HBV基因常见突变位点多态性 ,操作简便易行 ,技术要求不高 ,具有临床推广应用价值 ,而且可以方便地通过向寡核苷酸探针阵列中添加相应探针 ,扩大基因芯片的检测应用范围 ,为临床检测提供了新的方法     

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay.

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.