Alkaline Phosphatase-labeled Probe Research Articles

HPV 16 DNA and mRNA in cervical brush samples quantified by PCR and microwell hybridization

To quantitate HPV 16 DNA and mRNA, biotinylated amplicons from PCR and reverse transcription PCR were captured on streptavidin-coated microtitre plates. The amount of amplicon was determined by colorimetric detection after hybridization with an alkaline phosphatase-labelled probe. Dynamic ranges of between 4 and 6 log 10, sufficient to cover the amounts of viral DNA and mRNA prepared from cervical samples were achieved. The reproducibility of the colorimetric detection step was reflected in coefficients of variation (C.V.) below 8%, considerably better than that of chemiluminescense detection. In a series of 89 HPV 16 DNA positive cervical samples, as compared with a CIN I/normal diagnosis subgroup, the number of HPV 16 genome copies per assay was significantly greater in a CIN II subgroup ( P=0.014), and a high-grade neoplasia subgroup ( P=0.040), and the content of HPV 16 mRNA significantly greater in the high-grade neoplasia subgroup ( P=0.0021). The number of mRNA equivalents per copy of viral DNA was higher for E5 than for the other three mRNA species analyzed ( P<0.001), and the concentration of E6*I mRNA was higher than those of the E6 full-length ( P<0.001) and E6*II ( P<0.001) transcripts. Despite these differences, no correlation was found between histological/cytological diagnosis and the amount of viral mRNA relative to the viral load.

Detection of the infectious hematopoietic necrosis virus directly from infected fish tissues by dot blot hybridization with a non-radioactive probe

A method was developed for the rapid diagnosis of the infectious hematopoietic necrosis virus (IHNV) based on dot blot hybridization with a non-radioactive probe. When the assay was developed through a color reaction both biotin- and alkaline phosphatase-labeled probes were highly specific for IHNV and the sensitivity reached to 20 pg of viral RNA. When the alkaline phosphatase-labeled probe was developed by a chemiluminiscent reaction, the sensitivity showed a five-fold increase to 4 pg of viral RNA. The procedures successfully detected IHNV directly from infected symptomatic and asymptomatic fishes exhibiting higher sensitivity than the traditional approaches involving viral propagation in cell cultures. Additional advantages of the method are its simplicity and it takes only about 6 h to carry out the procedures from the initial processing of the tissues to diagnosis.

Patterns in expression of insulin-like growth factor-II and of proliferative activity in the normal human first and third trimester placenta demonstrated by non-isotopic in situ hybridization and immunohistochemical staining for MIB-1

The expression of insulin-like growth factor-11 (IGF-1I) in normal human first and third trimester placental tissue was investigated by non-isotopic in situ hybridization (ISH). This is the first ISH study on IGF-11 expression in placenta using an alkaline phosphatase-labelled probe. The expression was correlated with the proliferative activity of the cells using the proliferative marker MIB-1. In first trimester tissue, IGF-II was expressed in the cytotrophoblast, the extravillous trophoblast, the fetal endothelial cells and the mesenchymal fetal cells in the villi. In third trimester tissue, IGF-11 expression was found in the amnion, the extravillous trophoblast and the mesenchymal fetal cells especially in the endothelial cells and the outer contractile sheet in the stem villi. In areas with perivillous fibrin deposits, strong expression of IGF-11 was found in the cytotrophoblasts invading the fibrin. In first trimester tissue, the proliferative activity of the villous cytotrophoblast correlated well with the degree of IGF-II expression whereas in third trimester tissue, there was a discrepancy between MIB-1 positivity and the IGF-11 expression. Expression of IGF-II does not seem to be correlated exclusively to the mitogenic activity of cells.

Branched DNA for Quantification of Viral LOAD

This is a summary of a presentation made at the 13th International Convocation on Immunology. Nucleic acids in patient samples can be quantified directly using a solid phase nucleic acid hybridization assay based on branched DNA (bDNA) signal amplification technology. For example, HIV RNA is detected in a plasma sample by hybridization of multiple specific synthetic oligonucleotides to the target, 10 of which capture the target onto the surface of a microwell plate and 39 of which mediate hybridization of branched DNA molecules to the pol region of each HTV RNA molecule. Alkaline phosphatase-labeled probes bind to each arm of the branched DNA molecules. Detection is achieved by incubating the complex with a chemiluminescent substrate and measuring the light emission. The signal is directly proportional to the level of target nucleic acid, and the quantity of HIV RNA in a sample is determined by comparison with a 4-point standard curve. In order to ensure that different subtypes of HIV-1 were detected and quantified equally, in vitro RNA transcripts of the pol region of HIV subtypes A-F were purified and quantified by OD 260, phosphate analysis, and hyperchromicity. These characterized transcripts were then quantified using the bDNA assay. Comparisons were made using a ratio of signal per attomole for each transcript. Genetic subtypes A-F quantified within a factor of 1.5, indicating that the bDNA assay can be used to measure viral load in clinical samples regardless of genotype. Accuracy is important because several studies indicate that there may be a threshold level of virus which predicts progression of HIV disease. Detection of change in viral load is important in determining the efficacy of therapy. The bDNA assay for HCV RNA can be used to determine level of virus in HCV-infected individuals and assist in establishing prognosis prior to initiation of alpha-interferon therapy. Patients with lower levels of virus are more likely to have a sustained response to therapy. Patients who respond to treatment typically have a rapid decline in virus load within one to four weeks of the start of therapy. Many patients relapse when therapy is discontinued as evidenced by a rise in virus load to near pre-treatment levels. Sustained response is most often seen with patients who have lower pre- treatment levels of RNA.

Chemiluminescent enzyme immunoassay for detection of PCR-amplified enterotoxin A from Clostridium perfringens

A PCR protocol was developed for the rapid and specific detection of Clostridium perfringens strains harboring the enterotoxin A gene in artificially contaminated ground beef. A biotinylated primer pair was designed for amplification of a 750 bp fragment of the C. perfringens enterotoxin A gene. A combination of 4 h enrichment incubation and nucleic acid extraction, followed by 2 h of PCR amplification allowed detection at levels below 10 CFU of freshly grown cells in raw and cooked beef samples. PCR amplified products were confirmed by a Southern hybridization assay using a digoxigenin-labeled internal probe, and two hybridization ELISA protocols (PCR-ELISA) applying a streptavidin capture step for the hybridized PCR products. Both enzyme immunoassays utilized chemiluminescent detection with Lumiphos 530 ™ as substrate, after hybridization to an internal digoxigenin-labeled probe or a 5′ conjugated alkaline phosphatase-labeled probe. The PCR-ELISA resulted in faster confirmation of the PCR products while providing a level of sensitivity comparable to Southern hybridization, and has potential for development into an automated method.

Identified cholinergic neurones in the adult rat brain are enriched in GAP-43 mRNA: a double in situ hybridisation study

The cellular expression of growth associated protein-43 mRNA by identified choline acetyl transferase mRNA positive cells was investigated in the mature rat brain using a combined radioactive and non-radioactive in situ hybridisation technique. Cellular sites of growth associated protein-43 mRNA were detected using a 35S-oligonucleotide while choline acetyl transferase mRNA positive neurones were identified using two alkaline phosphatase-labelled probes. In the cholinergic cells of the corpus striatum, basal forebrain and laterodorsal tegmental nucleus a specific growth associated protein-43 hybridisation signal (silver grains) was detected, demonstrating that these choline acetyl transferase mRNA positive cells are enriched in growth associated protein-43 gene transcripts. By contrast, the large cholinergic cells of the motor nucleus of the trigeminal nerve did not express growth associated protein-43 mRNA. Quantification of the growth associated protein-43 hybridisation signal expressed by identified choline acetyl transferase mRNA positive cells showed regional variations in the relative cellular abundance of this transcript; cholinergic cells in the laterodorsal tegmental nucleus and corpus striatum expressed the strongest cellular hybridisation signal. Mean cross-sectional somatic area measurements of these growth associated protein-43/cholinergic positive cells confirmed the identity of these neurones as belonging to the cholinergic phenotype. A strong 35S-growth associated protein-43 hybridisation signal was detected also in numerous other non-choline acetyl transferase mRNA positive nerve cells in other regions of the brain, although the chemical phenotypes of these neurones were not determined. Our data reveal that expression of the growth-associated protein GAP-43 is maintained in identified cholinergic neurones in the postnatal rat brain, suggesting that this protein may subserve important functions in cholinergic and other neurones of the adult mammalian brain.

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Identification of Dengue virus-infected cells in paraffin-embedded tissue using in situ polymerase chain reaction and DNA hybridization

Using tissue from suckling mice infected with Dengue virus, the following improved method for detecting Dengue viral RNA in tissue sections was devised. Reverse transcription of the viral RNA to DNA was followed by the in situ polymerase chain reaction to amplify the viral nucleic acid. This was followed by DNA hybridization with an alkaline phosphatase-labelled probe. The enzyme was then reacted with Fast red and counterstained with hematoxylin. Viral nucleic acid was readily demonstrated within glial cells and macrophages in brains of animals infected with two different serotypes of Dengue.

Analysis of Trinucleotide Repeats in Myotonic Dystrophy

Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat in the 3-untranslated region of a gene encoding a serine-threonine protein kinase. There is no one method to detect the entire range of expansion sizes possible in affected patients, so current diagnostic approaches rely on analyzing samples by hybridization of both polymerase chain reaction (PCR)-amplified CTG repeats (CTG-PCR) and genomic DNA. In this unit, the the Basic Protocol 1 describes the analysis of PCR-amplified repeats transferred to a nylon membrane by Southern blotting and hybridized to an alkaline phosphatase-labeled probe. The first support protocol describes a vacuum blotting technique for rapid transfer of the PCR product to the nylon membrane and the second support protocol describes the use of a radiolabeled oligonucleotide probe for hybridization. Analysis of genomic DNA by similar hybridization techniques is outlined in the second basic protocol. Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat.

Fluorescent detection‐polymerase chain reaction (FD‐PCR) assay on microwell plates as a screening test for salmonellas in foods

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS200. The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55 degrees C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 microliters of 1 mmol 1(-1) AttoPhos (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1-10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR assay described here can be useful to screen a large number of food samples for contamination by salmonellas.

GAP-43 mRNA Expression in Facial Motoneurons During Regeneration: In situ Hybridization Histochemistry Study Using an Alkaline Phosphatase-labelled Probe

By means of in situ hybridization histochemistry using an alkaline phosphatase-labelled probe, we found an increase of mRNA for the growth-associated protein GAP-43 in rat facial motoneurons following axotomy of the facial nerve. After nerve resection, the increased level of GAP-43 mRNA was maintained for at least 8 weeks, while it returned to almost undetectable levels by 8 weeks after nerve crush injury. Thus, expression of GAP-43 mRNA in motor neurons paralleled the process of axonal regeneration. However, the increase of GAP-43 mRNA after resection was more pronounced than after crushing, in this way being different from the pattern of low-affinity nerve growth factor receptor mRNA expression.

Empirical evaluation of conditions influencing the polymerase chain reaction: enterotoxigenic Escherichia coli as a test case.

False polymerase chain reaction (PCR) results may be obtained under unfavourable reaction conditions. Therefore optimal conditions for the different factors influencing a specific PCR method should be determined before introduction to a clinical diagnostic laboratory. This study has concentrated on the detection of heat-labile enterotoxin-producing E. coli by PCR, with empirical evaluation of various factors. Template was prepared by heat-lysis of E. coli, and this was shown to be adequate for PCR detection. The results showed that deviation from the optimal conditions of any of the following conditions may lead to false results: lysis of bacterial cells, denaturation temperature during cycling, annealing temperature, primer concentration, enzyme concentration, magnesium concentration and ion concentration. Three different detection methods for PCR product were also evaluated. As little as one bacterium can be detected after 35 cycles of PCR amplification with 32P-labelled oligonucleotide probe. An alkaline phosphatase-labelled probe was 10-fold less sensitive, whereas 100 bacteria in 10 microliters of the original sample suspension were necessary to give a positive signal after gel electrophoresis. The information in this study may be useful to those who wish to introduce PCR tests to diagnostic laboratories.

Sensitive non-radioisotopic in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal

An alkaline phosphatase-labelled anti-sense oligonucleotide probe specific for tyrosine hydroxylase (TOH) mRNA has been used for visualisation of TOH mRNA in the rat brain and adrenal gland. Both ribonuclease pre-treatment and the use of excess non-labelled probe abolished the specific hybridization signal. Furthermore the TOH mRNA-positive signal was only found in cells known from earlier studies to react with anti-TOH antibodies. To determine if the alkaline phosphatase-labelled probe could be used in a semiquantitative manner for measurement of the density of TOH mRNA signal, we used reserpine pre-treatment which induces TOH mRNA expression. The results revealed a significant increase in TOH mRNA signal in locus coeruleus and substantia nigra neurons, and in adrenal medulla chromaffin cells. The increased signal in these areas agreed with the increase in TOH mRNA signal previously observed by Northern analysis and suggests that this type of alkaline phosphatase-labelled probe allows sensitive detection of changes in TOH gene expression.

Enteropathogenic Escherichia coli serotype O111:HNT isolated from preterm neonates in Nairobi, Kenya

This investigation was initiated as a consequence of several cases of diarrhea in a nursery ward for preterm babies in Nairobi, Kenya. Ten lactose-positive colonies were isolated from the stools of each of 30 neonates, regardless of whether they had diarrhea; 229 strains were identified as Escherichia coli and 65 strains were identified as Klebsiella pneumoniae. Six strains were lost during laboratory handling. No other bacterial, viral, or parasitic enteropathogens were identified. Using synthetic alkaline phosphatase-labeled probes, the bacterial isolates were found to be negative for the presence of genes coding for heat-stable and heat-labile enterotoxins. Seventy-eight E. coli strains isolated from a total of 13 neonates possessed the E. coli enteropathogenic adhesion factor (EAF) gene, as demonstrated by the use of a cloned radiolabeled DNA fragment probe. These strains possessed similar plasmid profiles constituting a core plasmid profile, and while all adhered to HeLa cells, none produced Vero cell cytotoxins. The EAF gene was located on a 65-megadalton plasmid. Serotyping showed the strains to be of serogroup O111 and serotype H nontypable, a well known enteropathogenic type. Five neonates died during the outbreak, and the fatality rate was 30.7% (4 of 13) for neonates infected with EAF-positive E. coli strains compared with 7.7% (1 of 13) for neonates from whom only EAF-negative E. coli strains were isolated. K. pneumoniae only was isolated from five neonates.