Genotyping of enteric adenoviruses by using single-stranded conformation polymorphism analysis and heteroduplex mobility assay

Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results.     

An heteroduplex mobility analysis assay based on capillary electrophoresis for the study of HCV quasispecies

The quasispecies nature of the hepatitis C virus (HCV) genome is central to the transmission, persistence and pathogenesis of the infection. Heteroduplex mobility analysis (HMA) is a simple and an inexpensive technique for the qualitative and quantitative analysis of genetic variation of viral quasispecies. An original HMA for the HVR1 region of HCV was developed, based on a semi-automated, non-radioactive capillary electrophoresis system, which allows the processing of large numbers of samples in short times, the accurate measure of mobility shifts and the quantitation of heteroduplexes. A set of 120 HVR1 clones of known sequence was used to develop the assay, which was tested on HVR1 sequences amplified directly from sera of 17 HCV-infected patients. HVR1 sequence divergence directly correlated with the heteroduplex mobility ratio (HMR) of hybrid molecules between six and 40 mismatches. Heteroduplexes between one and six mismatches were resolved, although HMRs were not proportional to base changes, likely due to an effect of type and position of the substitutions. The assay sensitivity was 1% of the total sample size. This assay may allow the application of quasispecies analysis to a wider range of clinical and basic investigations.     

Diversity of hepatitis C virus quasispecies evaluated by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intravenous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation between HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate variation in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not correlated with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.     

Investigating hepatitis C virus heterogeneity in a high prevalence setting using heteroduplex tracking analysis

Hepatitis C virus (HCV) infection is very common among chronic hemodialysis patients. In the past, blood transfusion appeared to be the primary risk factor; however evidence of nosocomial HCV transmission in the hemodialysis setting has recently been reported. This report describes a molecular investigation of HCV isolates obtained from a population of 670 patients attending six different Seattle-King County based hemodialysis centers in order to identify potential common source infections. 733 serum specimens were collected from hemodialysis patients in 1992 and 1996, and were tested for HCV antibodies and RNA. Overall, 115 of 670 (17%) patients were positive for HCV RNA, and thus were considered actively infected by HCV. HCV genotype was determined in all cases by restriction fragment length polymorphism, and 93 patients were found to be infected by HCV genotype 1. HCV envelope genes were amplified from the 93 patients with genotype 1 infection, and were studied in further detail by heteroduplex tracking analysis (HTA) using genotype 1a and 1b specific probes derived from the envelope 1 (E1) and envelope 2 (E2) genes. Genetic relatedness between pairs of HCV envelope genes was estimated by calculating the degree of gel shift relative to homoduplex controls. Nucleotide sequencing and phylogenetic analysis was used to confirm genetic relatedness detected by HTA. When HTA was performed using the E1 gene probe, 12 apparently related infections were detected; 10 of 12 (83%) of these infections were confirmed as truly related using the gold standard method of nucleotide sequencing plus phylogenetic analysis. Using an E2 gene probe, 24 infections were apparently related, but only six (25%) were confirmed by sequencing. As a control, 41 envelope genes, which were unrelated by HTA, were sequenced; 0 of 41 (0%) were truly related. In summary, HTA provides a rapid and effective molecular technique for screening HCV genetic relatedness in population-based studies, and should prove valuable in future studies of HCV molecular epidemiology.     

Genotyping of hepatitis C virus by Taqman real-time PCR.

BACKGROUND: Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries. OBJECTIVES: To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region. STUDY DESIGN: The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples. RESULTS: Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4. CONCLUSION: The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.     

Genotyping of Canadian hepatitis C virus isolates by PCR.

We used PCR for hepatitis C virus (HCV) genotyping with type-specific primers from the core and NS5 genes. Type I was predominant in the general population (58% in blood donors) as well as in different risk groups, such as intravenous drug abusers (58%), blood transfusion recipients (64%), hemophiliacs (62%), and patients with HCV chronic liver disease (76%). Types II, III, and IV were less prevalent in Canada, being found in 10.92, 6.72, and 5.88% of the population, respectively. The type II core primer was not type specific and reacted with the majority of our type I HCV samples, suggesting a false-positive dual infection with two different genotypes (I and II). Digestion of these amplified type I and type II products with restriction endonuclease AccI proved to be very useful in the exclusion of false-positive dual type I and type II infections.     

PCR-荧光探针法检测庆阳地区丙型肝炎病毒基因型

目的:应用PCR-荧光探针法分析庆阳地区丙型肝炎患者基因型,并对PCR-荧光探针法的各种性能进行评价。方法:收集庆阳地区289 例各种丙型肝炎患者的临床资料和外周静脉血,采用PCR-荧光探针法检测其基因型,并和PCR-反向点杂交法、测序法进行比较。结果:289 份HCV RNA 阳性血清标本中,PCR-荧光探针法基因型及亚型检出率为99.3%(287/289),其中1b 型139 例(48.1%),2a 型136 例(47.1%),3a 型7 例(2.4%),3b 型5 例(1.7%),未分出型2 例(0.7%)。PCR-荧光探针法的特异度和准确度为100%,重复性良好,并与PCR-反向点杂交法、巢式PCR 测序法分型结果均一致,三种方法的一致率为98.2%(56/57),差异无统计学意义(P>0.05)。1b 基因型患者ALT、AST、PLT 和HCVRNA(lg)水平均高于2a基因型患者,差异具有统计学意义(P<0.05)。结论:庆阳地区HCV 基因型呈现多基因型分布特点,主要为1b 型和2a 型,且2a 型和1b 型的比例相当,呈现出2a 型比例升高,1b 型下降的趋势;PCR-荧光探针法HCV 基因分型敏感度和特异度高,方法简单,适合临床实验中应用。     

Genotyping of hepatitis C virus in South Africa.

The six major hepatitis C virus genotypes were investigated by using samples from 79 seropositive and PCR-positive blood donors from three different regions of South Africa as well as 9 patients with chronic renal failure, 19 with liver disease, and 23 with hemophilia. PCR products of the genome were typed by restriction fragment length polymorphic analysis by RsaI-HaeIII and MvaI-HinfI double digestion. Type 5 occurred in 40% of this population group; type 1 occurred in 33%; and types 2, 3, and 4 were found in 13.8, 7.7, and 2.3%, respectively.     

应用非变性毛细管电泳检测乙肝病毒基因型B和基因型C

目的 建立非变性毛细管电泳分离乙肝病毒特异性巢式PCR产物方法,用于乙肝病毒B基因型和C基因型判断.方法 建立线性聚丙烯-聚乙二胺-TBE非变性毛细管电泳方法,分离母婴阻断失败患儿血清中乙肝病毒基因型特异性巢式PCR的扩增产物,采用表观分子量鉴定B、C基因型特异性和非特异性扩增产物.结果 毛细管电泳条件为30.2cm×50μm毛细管,分离缓冲液为2％(w/v)线性聚丙烯酰胺+0.4％(w/v)聚乙二醇+lxTBE缓冲液(pH8.3).在9kV电压下12分钟内可分离DNA分子量梯度标准品(50 bp至300 bp)及PCR扩增产物,表观分子量与电泳迁移时间的曲线适合度大于0.9999.B型扩增产物表观分子量范围为282bp至285bp,非特异性扩增产物为276bp至280bp.C型为ll9bp至120bp,当前扩增条件下不产生非特异性扩增产物.结论 非变性毛细管电泳能够方便和准确地判断非特异性的B型扩增产物.     

Human enterovirus isolates from an outbreak typed using heteroduplex mobility analysis

Genotyping and serotyping of enteroviruses is important for epidemiological, prognostic, and therapeutic reasons. In this study clinical isolates of enterovirus 71 during an outbreak of childhood meningoencephalitis in Sydney, Australia were identified using heteroduplex mobility analysis (HMA) of products from RT-PCR amplification of the 5' untranslated region. Five enterovirus 71 isolates shared identical heteroduplex patterns and nucleotide sequences in the 5' untranslated region. A sixth isolate exhibited minor differences in heteroduplex pattern and sequencing confirmed the isolate varied by 1% at the nucleotide level. The use of multiple reference strains and the analysis of heteroduplex patterns increased the confidence of isolate identification, and allowed identification of strain variation which could be subsequently further analyzed using sequencing. HMA can be used to accurately distinguish identical and variant isolates derived from sporadic cases and clustered infections with enteroviruses, including those causing serious infections.     

Mixed viral infection identified using heteroduplex mobility analysis (HMA)

It is now recognised that mixed viral infection, or infection of an individual with two or more distinct strains of a single viral species, often occurs particularly with RNA viruses. Current methods for detection of mixed infection normally involve genotyping or cloning and DNA sequencing. These methods are not always accurate or sensitive at detecting mixed infection and cannot be used for large numbers of samples. Furthermore subsequent sequence determination of the coinfecting viruses is labour intensive. This paper describes a simple, generic method based upon PCR and heteroduplex mobility analysis (HMA) that can be used to rapidly determine mixed infection with two strains of the same virus. The utility of this method is illustrated with hepatitis C virus (HCV) and TT virus (TTV) as examples. PCR-HMA detected mixed infection in 3 (8%) of 38 sera from intravenous drug users (IVDU) and 28 (30%) of 70 TTV-positive sera from Australia, China, and Vietnam. HMA can also be used to screen recombinant colonies to identify the sequences of the coinfecting viruses. The methods described here could be applied to analyse any PCR product containing two or more divergent sequences, whether derived from viruses, bacteria, or eukaryotic organisms.     

Differentiation of cassava-infecting begomoviruses using heteroduplex mobility assays

Cassava mosaic disease is an enormous problem affecting the viability and productivity of cassava in all the developing regions in Africa. The disease is now known to be caused by a number of cassava-infecting begomoviruses. New viruses and viral strains continue to be discovered and due to the lack of cloning and sequencing facilities in many African laboratories, a simple, rapid and sensitive technique is needed for screening of cassava plantations. Here we report on the development of a heteroduplex mobility assay (HMA) which could be used for the testing of viral-infected cassava. The assay involves amplifying the highly conserved core region of the coat protein gene of field isolates followed by denaturing and annealing with a number of reference strains. The HMA profiles in this study were able to differentiate four different viral species and 11 different virus strains, and showed a good correlation with sequencing results and phylogenetic comparisons with other sequenced cassava viruses. This technique is sensitive and rapid and has the added advantage of being able to detect mixtures of viruses in field-grown cassava.     

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Mutant K-ras provides an independent negative predictive marker for epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancers (CRCs). Rapid, sensitive, and cost-effective screening for K-ras status will overarch rational personalized medicine. Stool-based DNA testing offers unique advantages for CRC screening such as noninvasiveness, high specificity, and patient compliance, whereas complicated procedures and the low sensitivity of the present approaches have hampered its application on a wide scale. In this study, a chip-based temperature gradient capillary electrophoresis (TGCE) technique was applied to detect low-abundance K-ras mutations under a pooled experiment and analyze K-ras mutations in 30 paired stool samples and cancer tissues of CRC patients and 15 stool samples of healthy volunteers. The chip-based TGCE results showed that the successful analysis of K-ras status could be achieved within 6?min with an extremely low sample consumption of 14?nl. Detection is sensitive enough to reliably report 0.2% mutant CRC cells in a wild-type background, and 0.5?ng of template DNA was sufficient for chip-based TGCE. Of the 30 stool samples of CRC patients analyzed, 17 (57%) harbored K-ras mutations, and the lowest percentage of the detectable mutant K-ras in stool samples was 2%. The coincidence rate for K-ras mutations between stools and tissues obtained by the chip-based method reached 97% (29/30). One of the 15 stool samples of normal controls carried K-ras mutations, producing a specificity of 93%. Clone sequencing data entirely confirmed the results obtained by chip-based TGCE. The study demonstrates that chip-based TGCE is capable of rapidly screening low-abundance K-ras mutations with high sensitivity, reproducibility, simplicity, and significant savings of time and sample. Application of this method to genotype the K-ras gene in stools would provide a potential means for predicting the effectiveness of EGFR-targeted therapy in CRC patients using noninvasive approaches.     

Viral genome characterisation by the heteroduplex mobility and heteroduplex tracking assays

The heteroduplex mobility assay (HMA) is a means of comparing two PCR amplicons or, in the variation known as the heteroduplex tracking assay (HTA), a means of estimating the quasispecies diversity of a viral genome. Heteroduplex assays have many applications including subtyping viral genomes, screening for low frequency variants in a population, scanning the relative genetic diversity across a genome and screening for recombinant clones. They can be used to detect dual infections, superinfections, contaminated blood products and laboratory contaminations. PCR amplicons of about 65% sequence similarity or greater will form heteroduplexes under appropriate conditions, and phylogenetic trees can be drawn from heteroduplex mobility data. While homoduplexes indicate more than 98% similarity between two DNA sequences, heteroduplexes indicate at least seven mismatches in a 500-bp amplicon, or a three-base pair gap in 1000-bp. Minority variants comprising 1% to 5% of the genome population can be detected and quantified by HTA. Thus far, heteroduplex assays have been described for HIV and other lentiviruses, hepatitis C and G viruses, Norwalk-like viruses, influenza, measles and poliovirus. They could be applied to a wide range of other viral species.     

PCR结合单酶切对戊型肝炎病毒进行基因分型的研究

目的　建立PCR结合单酶切对戊型肝炎病毒(HEV)进行基因分型的方法,并将其应用于HEV基因型分布的研究。方法　采用简并引物扩增1～4型HEVORF1片段,1、2型HEVPCR产物为2 75、2 6 9bp ,3、4型PCR产物为317、314bp ,分别应用NaeⅠ、NotⅠ消化两种长度的PCR产物,根据酶切结果区分4种基因型。结果　采用此方法对4种基因型的HEV标准株分型,结果与预期一致;4 3份HEVIgM阳性的临床标本中,19份PCR阳性,分型结果均为4型HEV。结论　此方法可以快速简便地区分HEV 4种基因型。南京地区散发戊型肝炎大多由4型HEV所致。     

Genotyping and the quantification of hepatitis C virus by the melting curves in light cycler

Detection of the HCV genome is crucial for diagnosis of HCV infection and for monitoring the efficacy of interferon treatment for patients with HCV. We developed a convenient screening test for HCV genotypes 1 and 2 based on the melting curve analysis with SYBER green I. Serum samples were drawn from 114 patients with known chronic HCV infection confirmed to be antibody-positive by immunoblot assay. A characteristic melting profile for each genotype was obtained by monitoring the fluorescence as the temperature increases through the melting point of the PCR product. Serum samples with HCV-RNA genotype (1b, 2a and 2b) were analyzed every test as standard samples and the genotype of unknown samples was determined by the comparison with the melting point of standard samples. Serum samples with known HCV-RNA genotype (1b, 2a and 2b) and HCV-RNA-negative sample were tested using the Light cycler system. The melting curve analysis indicated that melting points are 93.08 +/- 0.56 degrees C for genotype 1b (n = 63), 91.08 +/- 0.49 degrees C for genotype 2a (n = 33), and 91.77 +/- 0.28 degrees C for genotype 2b (n = 18). The melting points for genotypes 1b, 2a, and 2b differed by approximately 1 degree C in each other. The genotype was determined for all samples using Okamoto's method and Light cycler system, and both systems produced absolutely identical results for all the samples studied. Sixty-three of 114 were genotype 1b, 33 samples were genotype 2a, and 18 were genotype 2b. This melting curve analysis is a rapid and convenient screening test for differentiation of HCV genotypes 1 and 2.