Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

Nonradioactive Detection of Small RNAs Using Digoxigenin-Labeled Probes.

Small RNAs have been traditionally detected and quantified using small RNA blots, a modified Northern blot technique. The small RNAs are size-fractionated from the rest of the cellular RNA molecules by polyacrylamide gel electrophoresis and transferred by blotting onto a positively charged membrane. A radiolabeled probe was then traditionally used to detect a specific small RNA in the cellular pool. Small RNA blotting is a relatively simple, inexpensive approach to visualize small RNAs without artifacts. However, the radioactive labeling of the probe is sometimes an impediment, especially due to the requirement of specialized facilities. Here we describe a sensitive and simple method to detect and quantify small RNAs using digoxigenin-based nonradioactive RNA blots.

Double-tagging polymerase chain reaction with a thiolated primer and electrochemical genosensing based on gold nanocomposite sensor for food safety.

A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. This assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection.

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100% agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100 percent agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

Simultaneous identification of orthopoxviruses and alphaviruses by oligonucleotide macroarray with special emphasis on detection of variola and Venezuelan equine encephalitis viruses.

The development of a method in macroarray format for the identification of alphaviruses and orthopoxviruses in samples of concern in biodefense is reported. Capture oligonucleotides designed to bind generic members of the orthopox- or alphavirus families and a collection of additional oligonucleotides to bind specifically nucleic acids from five individual alphaviruses, including Venezuelan equine encephalitis, or DNA from each of four orthopoxviruses, including variola virus (VAR) were deposited onto nylon membranes. Hybridization of digoxigenin labeled PCR products to the macroarray produced results easily observable to the naked eye. Multiplex RT-PCR utilizing both orthopox- and alphavirus-generic primers yielded amplification of DNA corresponding to the expected sizes of the orthopoxvirus and alphavirus fragments, respectively. Hybridization of samples to capture oligonucleotides in the macroarray membranes identified correctly generic orthopox- or alphaviral sequences. The hybridizations correctly identified each of the three alphaviruses and two orthopoxviruses tested. We observed cross-hybridization only once (between two alphaviruses) that was less intense than the spots formed by correct hybridization. The macroarray test described below is easy to perform, inexpensive, relatively fast, uncomplicated to interpret, and its end point is read visually without the need of additional equipment. This nucleic acid hybridization assay onto nylon membranes in macroarray format can help in detecting or excluding the presence of threat viruses in environmental samples and appears promising for a variety of biodefense applications.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies.

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4%), followed by genotype 3 (21.4%), and genotype 2 (7.2%). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4%), mild (57.2%), and moderate (21.4%). Viral RNA was detected in liver cells from nine patients (64.3%). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4 percent), followed by genotype 3 (21.4 percent), and genotype 2 (7.2 percent). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4 percent), mild (57.2 percent), and moderate (21.4 percent). Viral RNA was detected in liver cells from nine patients (64.3 percent). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA.

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.

Unidentified serogroups of enteropathogenic Escherichia coli (EPEC) associated with diarrhoea in infants in Londrina, Parana, Brazil.

Digoxigenin-labelled DNA probes were used to characterise enteropathogenic Escherichia coli (EPEC) isolated in Londrina (Brazil) from faeces samples of 102 children with diarrhoea, and the results were compared with those obtained by serogrouping and adherence to HEp-2 cells. The probes employed detect the gene coding EPEC adherence factor (EAF) and the virulence genes for bundle-forming pilus (bfp) and entero-attaching-effacing (eae) factor. Twenty-one isolates hybridised with at least one probe, and 11 of them were classified as typical EPEC because they hybridised with all three probes, showed a pattern of localised adherence (LA) and carried no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype 0119:H6 and one to O111:H6; O antigens could not be determined in four strains with antisera against 01-0173. All typical EPEC strains carried a 70-MDa plasmid plus two other large plasmids. These data showed that typical EPEC virulence traits may be found in strains not belonging to classical serogroups/serotypes and that molecular identification is required for studying the epidemiology of diarrhoea in children.

Detection of field isolates of human and animal group C rotavirus by reverse transcription-polymerase chain reaction and digoxigenin-labeled oligonucleotide probes.

Rotaviruses (RV) are important etiological agents of acute gastroenteritis in infants and young children, as well as the young of a variety of animals worldwide. These viruses belong to Reoviridae family and contain a genome of 11 segments of double-stranded RNA (dsRNA). Two major proteins, VP4 and VP7, encoded by genome segments 4 and 7, 8 or 9, respectively, evoke a neutralizing antibody response and form the basis for the current classification of group (gp) A rotavirus into P (VP4) and G (VP7) serotypes. Although much recent progress has been made on the molecular biology of gp C RV, routine methods to detect and discriminate human, porcine, and bovine strains are not available widely. In this study, a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and digoxigenin-labeled (dig) oligonucleotide probes using chemiluminescence has been developed to detect and discriminate VP7 genes from culture-adapted and field isolates of human, porcine and bovine gp C RV. The multiplex RT-PCR and dig-probes were specific for the VP7 genes of human, porcine and bovine gp C RV and allowed detection and characterization of single and mixed infections of porcine gp C RV with porcine gp A or gp B rotaviruses. Detection rates for gp C RV were more than 50% when compared with polyacrylamide gel electrophoresis. These new diagnostic assays may help determine the epidemiological importance of these viruses in human and animal infections.

In situ hybridization with digoxigenin-labelled RNA probes for detection procollagen 1 (I)

A localizaçao celular do pró-colágeno 1(I) foi observada na interface de coral natural, sete dias após implante no nosso trabecular de ratos, utilizando-se sondas de RNA marcadas com digoxigenina. A eficácia deste método foi comparada com aquela de sondas de RNA marcadas com enxofre(35). A hibridizaçäo "in situ" realizada utilizando-se sondas de RNA de osteoblastos, marcada com digoxigenina, provou ser täo eficiente quanto aquela marcada com S(35). Embora a porcentagem de fibroblastos marcados com S(35) tenha sido maior do que aqueles marcados com digoxigenina, näo houve diferença significante quanto à eficiência dos dois métodos

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

Evaluation of a colorimetric Babesia bigemina-DNA probe within an epidemiological survey.

An epidemiological survey was conducted in southeast Mexico, in an effort to establish the serological reactivity and carrier status to Babesia bigemina of an indigenous cattle population. The prevalence was obtained through the Indirect Fluorescent Antibody Test (IFAT), using an in vitro culture-derived B. bigemina antigen. A specific, digoxigenin-coupled, approximately 6 Kb B. bigemina-DNA probe (BBDP), was used to indicate the presence of the parasite. Serum samples from 925 animals of all ages, were obtained within the three regions (I, II, III) of the state of Yucatan and tested by IFAT. In addition, whole blood samples drawn from 136 of the same animals of region II were analyzed using the BBDP. Positive IFAT (IFAT+) reactions were observed in 531 sera for a 57% overall prevalence. Regional values were: I = 157+ (56%), II = 266+ (68%) and III 108+ (42%). Only 32 (23%) of the blood samples tested with BBDP showed distinctive hybridization signal, in contrast with 100 (73%) IFAT+ animals. The response distribution for IFAT vs. BBDP was: +/+ 23, +/- 77, -/+ 9 and -/- 27 respectively. It was found that the analytical sensitivity of BBDP appears to be low for its utilization in widespread epidemiological surveys. It was considered, however, that the colorimetric probe might be useful to safely detect transmission prone carriers, since it is able to detect parasitemias as low as 0.001%.

In situ hybridization with digoxigenin-labelled DNA probes for the detection of human papillomavirus-induced focal epithelial hyperplasia among Venezuelans.

An in situ hybridization assay with digoxigenin-labelled probes was used to detect the presence of human papillomavirus (HPV) sequences in ten related Venezuelan patients with the diagnosis of focal epithelial hyperplasia. The samples displayed HPV sequences in all cases. Further restriction analysis in four of the patients suggested the presence of HPV-13 in oral lesions.