Differentiation between wild and vaccine-derived strains of poliovirus by stringent microplate hybridization of PCR products.

Procedures for differentiation between wild and vaccine-derived strains of poliovirus are required, particularly in countries where wild and vaccine-related strains coexist. For this differentiation, we tested the method of Inouye and Hondo (S. Inouye and R. Hondo, Arch. Virol. 129:311-316, 1993) for discrimination of closely related viruses by using stringent microplate hybridization of PCR products. We used a pair of primers with enterovirus common sequences (between these primers there is a variable region for capsid proteins) for PCR using templates from wild and vaccine-derived poliovirus strains which were isolated in tissue culture and serotyped by neutralization assay. We also used the same primers for preparation of probes, which were labelled by incorporation of biotin-dUTP in the PCR, with the three original Sabin vaccine virus strains used as templates. The amplified DNAs from the isolates were immobilized on microplate wells and were then hybridized with the labelled probes. We found that, under the usual hybridization conditions, the Sabin vaccine virus strain probes hybridized with both wild and vaccine-derived viruses, but under stringent conditions, they reacted only with vaccine-derived viruses of the same serotype, clearly differentiating these from wild-type viruses.     

Genotyping of measles virus in clinical specimens on the basis of oligonucleotide microarray hybridization patterns

An oligonucleotide microarray hybridization method for identification of most known measles virus (MV) genotypes was developed. Like the conventional genotyping method, the microarray relied on detecting sequence differences in the 450-nucleotide region coding for the COOH-terminal 150 amino acids of the nucleoprotein (N). This region was amplified using PCR primers binding to all known MV genotypes. The microarray included 71 pairs of oligonucleotide probes (oligoprobes) immobilized on glass slides. Each pair consisted of a genotype-specific oligoprobe, which matched the sequence of only one target genotype, and a control oligoprobe, which contained mismatches at the nucleotide positions unique to this genotype. A pattern recognition algorithm based on cluster analysis of the ratios of hybridization signals from specific and control oligoprobes was used to identify the specific MV genotype. Following the initial validation, the method was used for rapid genotyping of two panels of coded samples. The results of this study showed good sensitivity (90.7%), specificity (100%), and genotype agreement (91.8%) for the new method compared to the results of genotyping conducted using phylogenetic analysis of viral sequences of the C terminus of the N gene. In addition, the microarray demonstrated the ability to identify potential new genotypes of MV based on the similarity of their hybridization patterns with those of known MV genotypes.     

基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。     

Detection and identification of Enterocytozoon bieneusi and Encephalitozoon species in stool and urine specimens by PCR and differential hybridization

Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. Enterocytozoon bieneusi and Encephalitozoon intestinalis are most commonly associated with chronic diarrhea. All Encephalitozoon species, including E. intestinalis, E. hellem, and E. cuniculi, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and Vittaforma corneae as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three Encephalitozoon species and V. corneae. An analytical sensitivity of 3.5 x 10(2) to 3.5 x 10(3) spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different Encephalitozoon species.     

Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization

Microarrays of virus-specific oligonucleotides may provide a method of screening samples for the presence or absence of a large variety of viruses simultaneously. Influenza viruses are ideal for evaluating such microarrays because of their genetic and host diversity, and the availability of an extensive sequence database. A collection of 476 influenza virus-specific oligonucleotides was spotted onto glass slides as probes. Viral RNAs were reverse transcribed and amplified by PCR, and the products were labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization. The fluorescence intensities of oligonucleotide spots were highly reproducible within each slide and satisfactorily proportional between experiments. However, the intensities of probe spots completely complementary to target sequences varied from background to saturation. The variations did not correlate with base composition, nucleotide sequence, or internal secondary structures. Therefore, thresholds for determining whether hybridization to a spot should be judged as positive were assigned individually. Considering only positive spots from probes predicted to be monospecific for influenza virus species, subtype, host source, or gene segment, this method made correct identifications at the species, hemagglutinin subtype, and gene segment levels. Monospecific neuraminidase (NA) subtype probes were insufficiently diverse to allow confident NA subtype assignment. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results. Overall, the results demonstrate the potential of microarray-based oligonucleotide hybridization for multiple virus detection.     

基因芯片技术检测环境中常见致病菌的初步研究

目的：为了快速，准确地检测环境中存在的致病菌，建立一种采用基因芯片技术对环境中常见致病检测和鉴定的实验方法。方法：采用合成后点样的方法把自行设计合成的一系列寡核苷酸探针固定在经过醛基化修饰的显微镜载玻片上，制成用于致病菌检测的基因芯片。结果：在相同的条件下，扩增了涉及12个菌属的151株细菌的165rDNA基因片段并与基因芯片杂交，经Scan-Array3000芯片阅读仪扫描得到特异性的交杂图，归纳这些杂交图，得到一套属（种）特异的典型杂交图谱。待检的样品菌与基因芯片进行杂交，得到的杂交结果与典型图谱比对即可判断出样品的种类。用这样的方法对从实际样品中分离的细菌进行检测，准确率表达了96．2％（25／26）。结论：该项技术的建立为今后更大规模的检测研究奠定了基础，可以推广应用于感染性疾病诊断，环境监测，食品卫生监督，商品检验检疫等领域。     

Identification of microsporidia in stool specimens by using PCR and restriction endonucleases.

We report the development of a PCR-based assay for the detection of microsporidia in clinical specimens. A single primer pair complementary to conserved sequences of the small-subunit rRNA enabled amplification of DNA from the four major microsporidian pathogens of humans: Encephalitozoon cuniculi, Encephalitozoon hellem, Enterocytozoon bieneusi, and Septata intestinalis. The extraction method allowed PCR amplification of E. bieneusi and S. intestinalis DNA from sodium hypochlorite-treated stool specimens. Differentiation of the microsporidian gastrointestinal pathogens E. bieneusi and S. intestinalis could be accomplished by restriction endonuclease digestion of PCR products using PstI and HaeIII.     

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.     

High-throughput identification of clinical pathogenic fungi by hybridization to an oligonucleotide microarray

Here we report the development of an oligonucleotide microarray method that can identify fungal pathogens in a single reaction. Specific oligonucleotide probes targeted to internal transcribed spacer 2 were designed and synthesized. Fungal DNA was amplified by universal primers, and the PCR product was hybridized with the oligonucleotide microarray. A series of specific hybridization profiles corresponding to species were obtained. The 122 strains of fungal pathogens, including standard and clinically isolated strains, used to test the specificity, stability, and sensitivity of the microarray system belonged to 20 species representing 8 genera. We found that the microarray system can successfully discriminate among the fungal pathogens to the species level, with high specificity and stability. The sensitivity was 15 pg/ml of DNA. This oligonucleotide microarray system represents a rapid, simple, and reliable alternative to conventional methods of identifying common clinical fungal isolates.     

Specific detection and typing of adenovirus types 40 and 41 in stool specimens by dot-blot hybridization.

A dot-blot hybridization test was developed which allowed the direct detection of fastidious enteric adenovirus DNA in stool specimens from children with diarrhea and simultaneous typing of the viruses as adenovirus type 40 (Ad40) or Ad41. Cloned PstI fragments of Ad40 and Ad41 were used as 32P-labeled probes in the test, which allowed detection of picogram quantities of viral DNA in 20 to 40 microliter of stool suspension. Results were obtained within 48 h. The type specificity of the test was evaluated with 76 specimens known to contain either Ad40 or Ad41 by restriction enzyme analysis. Sixty-one specimens had sufficient DNA to be detected without any removal of protein. Thirty-one adenoviruses were typed as Ad40, and 30 were typed as Ad41, giving 100% correlation with the results of restriction enzyme analysis. The other 15 specimens were detected and typed as Ad40 or Ad41 only after removal of protein by a phenol extraction method. The dot-blot hybridization method is particularly useful for identifying those Ad40 and Ad41 strains which defy all attempts at culture and will be a useful tool in the epidemiology of fastidious enteric adenovirus infections.     

Specific detection of toxigenic strains of Clostridium difficile in stool specimens.

Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone.     

Detection of genital human papillomavirus by single-tube nested PCR and type-specific oligonucleotide hybridization.

Cervical cancer is, on a global scale, the second most common form of cancer in women. Development of cervical carcinoma is strongly associated with infection by certain types of human papillomavirus (HPV). To facilitate the detection and molecular typing of HPV in clinical samples, nested-PCR amplification systems were developed for regions of the E1 and L1 genes. The nested amplifications were performed in a single reaction tube, and shifting between inner and outer primer pairs was achieved by a two-phase amplification with different annealing temperatures. This method eliminates cross-contamination between samples during transfer from the first to the second amplification step. A set of type-specific oligonucleotide probes were designed for the E1 system and used to distinguish 19 genital HPV types. The sensitivities of our amplification systems compare favorably with that for the L1 system on the basis of the MY09-MY11 primer pair (M.M. Manos, Y. Ting, D. K. Wright, A. J. Lewis, T. R. Broker, and S. M. Wolinsky, Cancer Cells 7:209-214, 1989) and our systems can be used on materials such as HPV-infected cell lines, cytobrush samples, cancer biopsies, and recent as well as archival Papanicolaou (Pap) smears. The high sensitivity coupled with the effective elimination of contamination in the transfer between the two amplification steps of the nested PCR makes these systems suitable for research as well as clinical analyses.     

Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR.

In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V. cholerae O139 Bengal. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR.     

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.     

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.     

Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens

Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.