Thermal degradation of RNA-RNA hybrids during hybridization in solution.

Degradation of single-stranded RNA molecules at high temperatures was examined in relation to the kinetics of RNA-RNA hybridization in solution. Eleven species (ranging from 670 bases to 3300 bases) of single-stranded RNAs transcribed from rotavirus genomic RNAs degraded significantly after 16 h of incubation at 65 degrees C. The hybridization of these 11 RNA molecules to the corresponding genomic RNAs, however, was completed within 30 min of incubation. Partially homologous hybrids that were once formed at an early time of incubation gradually degraded in proportion to the length of incubation at 65 degrees C. Thus, the length of hybridization has a critical effect on the final hybridization results. Furthermore, thermal hydrolysis of single-stranded RNA molecules provides a plausible explanation why the percent of nucleotide sequence mismatch allowed to form a stable hybrid in the RNA-RNA hybridization assays for rotavirus genes is much less than that predicted by calculation.     

Identification of rotavirus particle types

Negative-contrast electron microscopy of purified rotavirus particles reveals two particle types: single-shelled and double-shelled particles. The relationship of these particle types, seen by negative staining, to the enveloped and various types of nonenveloped particles seen in thin sections of virus-infected cells was determined. Thin-section and negative-contrast electron microscopic analyses were performed on cell lysates from simian rotavirus. SA11-infected cells and on highly purified double- and single-shelled particles. In thin sections, double-shelled particles appeared as smooth-edged ovals containing dense nucleoids, whereas single-shelled particles had ragged edges and threads of material extending from their centers. The majority of nonenveloped particles seen in thin sections of infected cells were identified as double-shelled particles. Enveloped particles showed typical membrane structure and were observed rarely in crude rotavirus stocks, although they constitute about 10% of the particles within infected cells. It is hypothesized that the enveloped form is a transient one and the envelope is lost in the endoplasmic reticulum of the host cells. Finally, the 50-55 nm type IV particles seen within lysosome-like bodies in infected cells were identified as subviral particles formed from input virions.     

Identification of Bacillus anthracis by multiprobe microarray hybridization

We have developed a rapid assay based on microarray analysis of amplified genetic markers for reliable identification of Bacillus anthracis and its discrimination from other closely related bacterial species of the Bacillus cereus group. By combining polymerase chain reaction (PCR) amplification of six B. anthracis-specific genes (plasmid-associated genes encoding virulence factors (cyaA, pagA, lef, and capA, capB, capC) and one chromosomal marker BA-5449) with analysis of amplicons by microarray hybridization, we were able to unambiguously identify and discriminate B. anthracis among other closely related species. Bacillus identification relied on hybridization with multiple individual microarray oligonucleotide probes (oligoprobes) specific to each target B. anthracis gene. Evaluation of the assay was conducted using several B. anthracis strains (with or without pXO1 and pXO2 plasmids) as well as over 50 other species phylogenetically related to B. anthracis, including B. cereus, B. thuringiensis, B. mycoides, and B. subtilis. The developed microarray analysis of amplified genetic markers protocol provides an efficient method for (i) unambiguous identification and discrimination of B. anthracis from other Bacillus species and (ii) distinguishing between plasmid-containing and plasmid-free Bacillus anthracis strains.     

Improved detection of rotavirus RNA in dot-blot hybridization assay by chromatographic extraction and acid denaturation of double-stranded RNA

One of the commonest methods of denaturing nucleic acids, denaturation by heat, was found to be ineffective for double-stranded (ds)RNA when RNA preparations contain 200 mM or higher concentrations of NaCl. We report acid denaturation of dsRNA (incubation for 10 min in the presence of 80 mM HCl) was particularly useful for such preparations. When genomic dsRNAs of rotavirus were extracted by ion-exchange chromatography columns (Extractor) and then denatured by acid, the detection of rotavirus RNA in stool specimens by the previously reported dot-blot hybridization assay (Yamakawa, K. et al. (1989). Molecular and Cellular Probes 3, 397-401.) was significantly improved.     

应用抑制性消减杂交技术克隆和筛选白血病多药耐药相关基因

目的 克隆和筛选白血病多药耐药（MDR）相关基因。方法 以MDR白血病细胞株K562/DOX为检测样本。以K562细胞株为驱赶样本（对照）。应用抑制性消减杂交（SSH）技术分离和克隆K562/DOX细胞中过高表达的基因。构建cDNA消减库；通过反向Northern斑点杂交法进一步筛选消减库，对阳性克隆进行序列测定和同源性分析；并采用RT-PCR和Northern blot方法对某些阳性克隆进行进一步的验证。结果 发现了11个在K562/DOX细胞中高表达的基因。包括S3核糖体蛋白（S3ribosomal protein,S3rp)基因，尼克酰胺腺嘌呤二核苷酸脱氢酶亚单位2（NADHdehydrogenase subunit2,ND2）基因，My023基因等，其中多数基因与肿瘤MDR的关系尚未见报道。结论 筛选到几个可能与白血病MDR形成有关的基因。提示了白血病MDR发生的新机制。     

Identification and genotyping of Norwalk virus by stringent microplate hybridization of PCR products

There are many genotypes of Norwalk virus(NV), and the microplate hybridization method for identification and genotyping of NV requires preparation of many probes. However, the method is simple and the detection sensitivity is high at 10 ng/ml DNA, and many samples can be simultaneously examined in an identification test and genotyping of NV. Increasing the hybridization temperature increases precision and the homology rate between a probe and DNA fragment can be estimated. Mixed genotypes of NV are often detected in a same sample of NV-infected patients and oysters. In such cases, genotypes cannot be determined by direct sequencing. However, this method is applicable to genotyping of cases infected with mixed genotypes. Genotypes can be determined without sequencing by preparing many probes.     

Identification of rotaviruses by dot-blot hybridization using an alkaline phosphatase-conjugated synthetic oligonucleotide probe

We have evaluated a recently-developed dot-blot hybridization assay for the detection of human rotaviruses using an alkaline-phosphatase conjugated oligonucleotide probe. The lower detection limit of this assay was 1 ng (approximately 5 x 10(7) copies) of the double-stranded (ds) RNA, when a purified preparation from serotype 1 human rotavirus was used but appeared to be much higher when applied on clinical specimens. This assay could detect dsRNA from rotavirus strains belonging to serotypes 1 through 6, 8 and 9. A total of 235 stool specimens were used to evaluate the oligonucleotide probe assay in comparison with polyacrylamide gel electrophoresis and latex agglutination assay (Rotalex). When polyacrylamide gel electrophoresis was used as a gold standard, sensitivity, specificity and positive and negative predictive values of the probe assay were 84, 100, 100 and 89%, respectively. These values were slightly better than those of Rotalex assay which is commonly used in clinical laboratories in Japan. Although the probe assay requires more hands-on time than the immunoassays, the high specificity of this probe assay recommends it as a confirmatory test in the clinical laboratory setting.     

Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes.     

Identification of rare Candida species and other yeasts by polymerase chain reaction and slot blot hybridization

Invasive candidiasis has become a major cause of morbidity and mortality in immunocompromised hosts. Here we describe a fast and reliable DNA extraction and PCR amplification method in combination with a slot blot hybridization assay. A genus-specific probe was designed that allowed to detect DNA from a broad range of Candida species and 3 other yeasts. In addition, species-specific oligonucleotides for emerging Candida and other yeast species allowed to identify DNA extracted from Candida lusitaniae, Candida humicola, Candida kefyr, Candida inconspicua, Candida solani, Malassezia furfur and Trichosporon cutaneum. A sensitivity of at least 10(1) CFU, corresponding to 100 fg of fungal DNA, was documented for all species-specific probes and the common Candida probe. In addition, the 18S rRNA genes of 7 yeast species (C. humicola, C. kefyr, C. solani, C. inconspicua, C. norvegensis, C. utilis and M. furfur) were completely sequenced. The sequencing primers described bind to highly conserved primer binding sites. Therefore, these primers would allow rapid cycle sequence of additional ribosomal genes throughout the whole kingdom of fungi.     

Impact of rotavirus vaccines on rotavirus disease

Rotaviruses are the most common cause of acute gastroenteritis in young children worldwide. Both licensed rotavirus vaccines (Rotarix? [RV1] and RotaTeq? [RV5]) are effective and safe. Studies from countries that have included RV1 or RV5 in the national immunization programs have demonstrated their safety and sustained efficacy under real-life circumstances. A significant decline in acute gastroenteritis-related deaths among Latin American children was observed after the introduction of RV1 and RV5 vaccines. Both vaccines were able to decrease the number of cases of rotavirus acute gastroenteritis and of severe rotavirus diseases. Vaccination was also associated with a dramatic reduction in hospitalizations and outpatient visits for all-cause acute gastroenteritis. Indirect protection after infant mass vaccination has been strongly suggested. Moreover, postlicensure safety studies assessed rare adverse events (rates <1 in 50,000), such as intussusception.     

Impact of rotavirus vaccines on rotavirus disease

Rotaviruses are the most common cause of acute gastroenteritis in young children worldwide. Both licensed rotavirus vaccines (Rotarix? [RV1] and RotaTeq? [RV5]) are effective and safe. Studies from countries that have included RV1 or RV5 in the national immunization programs have demonstrated their safety and sustained efficacy under real-life circumstances. A significant decline in acute gastroenteritis-related deaths among Latin American children was observed after the introduction of RV1 and RV5 vaccines. Both vaccines were able to decrease the number of cases of rotavirus acute gastroenteritis and of severe rotavirus diseases. Vaccination was also associated with a dramatic reduction in hospitalizations and outpatient visits for all-cause acute gastroenteritis. Indirect protection after infant mass vaccination has been strongly suggested. Moreover, postlicensure safety studies assessed rare adverse events (rates <1 in 50,000), such as intussusception.     

Identification of HIV-1 infected seropositive subjects by a direct diagnostic test involving hybridization of amplified viral DNA

We describe a simple slot-blot hybridization procedure with an oligo-probe for identification of amplified HIV-1 DNA in seropositive subjects. Comparison are realized, on the same DNA samples, with other methods DNA detection including Southern blot tests after hybridization with a labelled probe, and autoradiographic patterns after digestion with a restriction enzyme of the amplified product hybridized. This new direct approach towards diagnosis of HIV-1 infection easily be carried out on a large scale.     

Identification of DNA homologies among H incompatibility group plasmids by restriction enzyme digestion and Southern transfer hybridization.

Plasmids belonging to the three HI plasmid incompatibility subgroups were characterized by the use of restriction enzymes and Southern transfer hybridization. A diversity of restriction enzyme patterns was noted among the HI subgroups, and a small amount of DNA homology was observed by probing these digests with a nick-translated HI1 plasmid. Within a single subgroup (HI1 and HI2), similar restriction enzyme patterns were noted. Plasmids of all three HI subgroups and the HII group had a guanine plus cytosine content of 49 to 50 mol%. The IncHII plasmid pHH1508a also showed some homology with the HI1 probe. The DNA homology observed is probably responsible for common phenotypic properties encoded by these plasmids.     

Identification of Aeromonas trota (hybridization group 13) by amplification of the aerolysin gene using polymerase chain reaction

Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors. Rapid detection and identification of A. trota is important for early and specific diagnosis of the infectious diseases that it causes. Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A. trota. A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A. trota. The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments. The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1. Primer specificity for A. trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups. A strain of Aeromonas enteropelogenes that had been reclassified as A. trota was also PCR positive. The method described here can be used to detect aerolysin-producing A. trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests.     

常规细胞遗传学检查联合多重荧光原位杂交技术确定急性白血病患者复杂核型异常

目的 探讨多重荧光原位杂交(M-FISH)技术在急性白血病(AL)患者复杂核型异常和标记染色体检测中的应用价值.方法 对11例常规R显带检测显示具有复杂核型异常和标记染色体的AL患者应用M-FISH技术确定复杂染色体重排和标记染色体的组成,识别并确定微小易位和隐匿易位.结果 11例AL患者应用常规细胞遗传学(CC)技术共检出27种染色体数目异常和41种结构异常.CC技术检出的3种染色体增加和9种染色体丢失以及12种结构异常与M-FISH分析结果一致,CC技术检出的15种染色体丢失经M-FISH证实为衍生染色体,M-FISH还检出3种CC检查未发现的染色体数目增加.CC检查所检出的其余29种结构异常(包括17种标记染色体)的性质被M-FISH进一步明确.M-FISH共检出33种结构重排,有6种异常未见文献报道,分别为t(5q-;16)(?q14;q24)、der(9)(Y::9::Y::9)、der(7)(7::8::9)、ins(20;21)、der(11)(11::21::20)和der(3)t(3p-;13)(3p-;q21),这些复杂染色体重排主要由染色体不平衡易位所致.复杂核型异常几乎涉及所有染色体,在AML患者以涉及17、5、7、15和11号染色体的异常较为常见;在ALL患者则以涉及8、9、14和22号染色体的异常较为多见.结论 联合应用CC和M-FISH检查可以提高CC核型分析的分辨率,对伴复杂核型和标记染色体的AL具有一定的临床应用价值.     

Nucleic acid analysis by sandwich hybridization

One of the most significant achievements of the biochemist during the past two decades is the use to which immunologically based assays have been put in clinical diagnosis (Hood et al.: Immunology, 1984). The problem faced and surmounted by immunologists in effecting the transition from research tool to routine clinical assay bears a remarkable similarity to that confronting the molecular biologist today; i.e., how can nucleic acid hybridization, a technique of obvious potential (Meinkoth and Wahl: Anal Biochem 138:267-284, 1984; Syvanen: Med Biol 64:313-324, 1986; Matthews and Kricka: Anal Biochem 169:1-25, 1988), be modified in order to fulfill all necessary parameters of a routine diagnostic assay? There are several such requirements, and the importance placed on each depends on the objectives of the assay: the technique must be sensitive, specific, and reproducible. Other advantages would be cost-effectiveness, ease of manipulation, and amenability to automation. Ideally, the signal detection should be based on a non-radioactive system, because of the instability of probes labelled with isotopes like 32p, and the potential hazards involved in their handling and disposal. The sandwich hybridization for the analysis of nucleic acid sequences was first used in 1977 (Dunn and Hassell: Cell 12:23-36, 1977), but its potential as a diagnostic assay was not realized until 1983, when it was applied to the detection of adenovirus DNA in nasopharyngeal aspirates from children with acute respiratory infection (Ranki et al: Gene 21:77-85, 1983). It has since been modified and used not only for the detection of microbial infection (Virtanen et al.: Lancet i:381-383, 1983; Ranki et al.: Cur Top Microbiol Immunol 104:307-318, 1983; Lehtomaki et al.: J Clin Microbiol 24:108-111, 1986; Virtanen et al.: J Clin Microbiol 20:1083-1088, 1984; Palva and Ranki: Clin Lab Med 5:475-490, 1985; Polsky-Cynkin et al.: Clin Chem 31:1438-1443, 1985; Parkkinen et al.: J Med Virol 20:279-288, 1986; Palva: FEMS Microbiol Lett 28:85-91, 1985; Palva et al: FEMS Microbiol Lett 23:83-89, 1984; Zolg et al.: Mol Biochem Parasitol 22:145-151, 1987; Palva: J Clin Microbiol 18:92-100, 1983), but also for the analysis of nucleotide sequence variations (Langdale and Malcolm: Gene 36:201-210, 1985). We will discuss the development of the sandwich technique and the advantages it conveys over the more conventional nucleic acid hybridization formats, together with new developments which will ensure that it earns a place alongside immunoassay in the diagnostic laboratory.     

Microbial diagnostics by nucleic acid hybridization

Recombinant DNA techniques have enabled the selection and production of any specific gene and thus allowed the development of nucleic acid hybridization techniques as diagnostic tests for microbial identification. In the hybridization reaction sample nucleic acids, rendered single stranded by denaturation, are allowed to anneal with known specific probes carrying isotopic or nonisotopic label thus enabling the homologous target sequences in samples to be identified. Several hybridization tests have been established and used for the detection of many subviral, viral, bacterial and eukaryotic pathogens, which are difficult to diagnose by other methods. The most important challenge now for the hybridization techniques is in the development of practical tests, simply and rapidly performed, with high sensitivity, automation and nonisotopic detection system.