Alkaline Phosphatase-labelled Oligonucleotide Probe Research Articles

Evaluation of an alkaline phosphatase-labeled oligonucleotide probe for detection and enumeration of vibrio spp. from shrimp hatchery environment

An alkaline phosphatase (AP)-labeled genus-specific oligonucleotide probe was developed to detect and enumerate vibrios in shrimp larvae and their surrounding environment. The probe was evaluated using 35 laboratory isolates of Vibrio species and 29 isolates of non-vibrio species. The probe was specific for the Vibrio species and no cross reaction was found with the non-vibrios included in the study. The total Vibrio counts obtained by plating on thiosulfate-citrate-bile salts-sucrose agar (TCBS) and using oligonucleotide probe were compared. Total Vibrio counts obtained by probe were comparatively higher than the counts obtained by plating on TCBS agar. The difference between the counts obtained by the probe and by plating on TCBS agar ranged from 2 to 21 times. The study reveals that the use of a non-selective medium such as T1N3 agar followed by detection using a genus-specific probe would help to precisely enumerate the total Vibrio load in the aquaculture environments.

Evaluation of an Alkaline Phosphatase–Labeled Oligonucleotide Probe for the Detection and Enumeration of the Thermostable-Related Hemolysin (trh) Gene of Vibrio parahaemolyticus

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase–labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.

Evaluation of an alkaline phosphatase-labeled DNA probe for enumeration of Vibrio vulnificus in Gulf Coast oysters

A direct plating method and an FDA method were compared for enumeration of Vibrio vulnificus in Gulf Coast oysters. The direct plating method was based on hybridization of colony lifts and used an alkaline phosphatase-labeled oligonucleotide probe that targeted the cytolysin gene (Direct-VVAP). The FDA method employs most probable number analysis with confirmation of suspect isolates by enzyme immunoassay (MPN-EIA). Indigenous V. vulnificus levels in oysters harvested from Florida, Alabama and Louisiana throughout the year and in Gulf Coast market oysters ranged from <10 to 10 5 g −1. Similar V. vulnificus levels ( r=0.66) were found with these two procedures in freshly harvested oysters collected between April and October. Measurement variance was much greater, however, with the MPN procedure (0.118) than with the DNA probe procedure (0.004). In market oysters, the MPN-EIA procedure gave 0.4 log 10 higher estimates than the Direct-VVAP method, but the methods were closely correlated ( r=0.83). Confirmation procedures were in agreement >90% of the time, as suspect isolates confirmed by one procedure were identified by the other procedure. Except when V. vulnificus densities are low (<10 g −1), the Direct-VVAP method provides an alternative procedure that is more rapid and precise than the MPN-EIA analysis.

Dexamethasone enhances level of GAP-43 mRNA after nerve injury and facilitates re-projection of the hypoglossal nerve.

Application of dexamethasone was found to induce an enhanced expression level of mRNA encoding the growth associated protein (GAP-43) after peripheral nerve injury. Following hypoglossal nerve axotomy, a dexamethasone releasing pellet (1.5 mg released in 3 weeks) was placed near the transected nerve. GAP-43 mRNA was detected in the hypoglossal nucleus by non-radioactive in situ hybridization histochemistry using an alkaline phosphatase-labeled oligonucleotide probe. A significant elevation of GAP-43 mRNA level was observed 2 weeks after the transection in dexamethasone treated animals. This induction was not observed in the dorsal motor nucleus of vagus which expresses moderately high levels of GAP-43 mRNA even without nerve injury. Although dexamethasone did not alter the maximum level of GAP-43 mRNA in the hypoglossal nucleus after nerve injury, it prolonged the period in which the mRNA expression remained elevated. This may be due to post-transcriptional effect by the glucocorticoid. Dexamethasone treatment also caused a slight facilitation of reprojection. This may be due to the enhancement of GAP-43 mRNA level by the glucocorticoid.

Colchicine induces the GAP-43 gene expression in rat hypothalamus.

Injection of colchicine, a mitogen inhibitor, in the dorsal third ventricle induced the expression of the growth associated protein-43 (GAP-43) mRNA in some groups of cells of the adult rat brain. These mRNAs were detected by in situ hybridization histochemistry using an alkaline phosphatase labeled oligonucleotide probe. A substantial up-regulation of GAP-43 mRNA was noticed by the increase of both the number of positive cells and the intensity of the hybridization signal. These changes were observed in the hypothalamic nuclei located near the ventral third ventricle, namely the preoptic area, the supraoptic nucleus, the peri- and the paraventricular nuclei of the hypothalamus, the dorsal subnucleus of the ventromedial nucleus, the arcuate nucleus and the posterior part of the peri-mammillary region. Such abundant GAP-43 mRNA positive cells have not been observed in control adult rat hypothalamus. Since the positive cell number and shape initially suggested that these were neurons or astrocytes, double labeling in situ hybridization using both radioactive (for the detection of GFAP mRNA as a marker of astrocyte) and non-radioactive (for the detection of GAP-43 mRNA) probes was carried out. This demonstrated that these GAP-43 mRNA positive cells were not astrocytes. In addition enhanced GAP-43 mRNA expression was also found in some neuronal component, particularly in neurosecretory magnocells of the pareaventricular and the supraoptic nuclei. This up-regulation was further confirmed by the Northern blot analysis. About five fold increase in GAP-43 mRNA in the colchicine-treated hypothalamic tissue was shown.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of calretinin mRNA in the vestibular nuclei of rat and guinea pig and the effects of unilateral labyrinthectomy: a non-radioactive in situ hybridization study

We examined the localization of neurons expressing mRNA for calretinin, a cytosolic EF hand calcium-binding protein, throughout the vestibular nuclei of rat and guinea pig by non-radioactive in situ hybridization, using an alkaline phosphatase labeled oligonucleotide probe. Labeled cells were particularly numerous in the medial vestibular nucleus (mVN) and their distribution was similar in rat and guinea pig, and presented a characteristic rostrocaudal and mediolateral pattern. The effects of hemilabyrinthectomy were assessed at various times post lesion from 10 h to 30 days by comparison of the pattern of labeling in the ipsi- and contra-lateral vestibular nuclei of guinea pig. After up to 48 h no modification in the calretinin mRNA distribution was detected. After 3 to 30 days of survival, there was a decrease (about 30%) of the calretinin expressing neurons in the nucleus on the side of the lesion. The unilateral sensory deprivation seemed to induce a permanent asymmetry in the expression of calretinin which was not abolished after vestibular compensation. These results suggested that the calretinin expression in these neurons depends upon the integrity and activity of sensorineuronal peripheral vestibular influences.

Optimisation and evaluation of a quantitative chemiluminescent polymerase chain reaction assay for hepatitis C virus RNA.

A quantitative non-isotopic assay for measuring hepatitis C virus (HCV) RNA has been developed and evaluated. Viral RNA extracted from serum is reverse transcribed and amplified by the polymerase chain reaction (PCR) using biotinylated 5' non-coding region primers. PCR products are captured on streptavidin coated microtitre plates, denatured with sodium hydroxide and hybridised with an alkaline phosphatase-labelled oligonucleotide probe. Quantification is achieved by measuring the intensity of light emitted by a dioxetane-based chemiluminescent substrate. The chief advantages of the assay are: (i) extreme sensitivity with the ability to detect single molecules of HCV cDNA, (ii) a 5 log10 dynamic range sufficient to cover the 10(3)-10(8) genomes/ml viraemia levels typically seen in patient samples, (iii) specificity and reproducibility suitable for application in a clinical context, and (iv) a rapid non-nested assay format with the ability to handle large throughputs and with a potential for automation. The feasibility of using the assay to monitor viraemia level changes in patients undergoing interferon therapy for chronic HCV infection has been demonstrated.

Neurotensin transcription is oppositely regulated during development and regeneration.

Our previous work demonstrated that neurotensin mRNA expression was transiently up-regulated during development in the hypoglossal motoneurone. The present study was carried out to examine how neurotensin mRNA expression changes during nerve regeneration. Following either unilateral resection or crushing of the hypoglossal nerve, mRNA expression was detected by non-radioactive in situ hybridization histochemistry using an alkaline phosphatase-labelled oligonucleotide probe. A reduction in neurotensin mRNA was observed on the operated side after the nerve injury. It took around 2 months for NT mRNA levels to return to normal. Similar down-regulation was observed following crushing, but recovery was more rapid than that observed with sectioning, probably due to the extent of nerve damage. This finding indicates that neurotensin transcription is up-regulated during development but, on the contrary, down-regulated during regeneration.

Detection and occurrence of waterborne bacterial and viral pathogens

Detection and occurrence of waterborne bacterial and viral pathogens

Evaluation of alkaline phosphatase-labelled ipaH probe for diagnosis of Shigella infections.

The presence of many enteropathogens which are not easily detectable by routine stool culture has led to the development of alternative diagnostic methods. One of these techniques, nucleic acid probe hybridization, has been used to identify Shigella spp. and enteroinvasive Escherichia coli (EIEC) in stool specimens through the detection of genetic material encoded by a specific large approximately 200-kbp virulence-related plasmid. In the present study, an alkaline phosphatase-labelled oligonucleotide probe developed to detect the gene for ipaH, a repetitive genetic sequence thought to be present on both the virulence-related plasmid and the chromosomes of all strains of Shigella and EIEC, was tested in a developing-country setting through a prospective clinical trial. In a group of 219 Peruvian adults and children with acute gastroenteritis, the ipaH probe detected 85% of cases of shigellosis and demonstrated a specificity of 95% when compared with simultaneous detection by several stool culture techniques. Additionally, three cases of EIEC infection which could not be diagnosed by culture methods alone were detected with the ipaH probe and were confirmed by plasmid analysis and Sereny testing. These preliminary results suggest that, with further research, the ipaH probe should prove to be a useful and rapid adjunct in the diagnosis of acute gastroenteritis in developing countries.

Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe.

An oligonucleotide DNA probe (VVAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hylA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 10(2) and 2 x 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests.

Contrasting Effects of Raclopride and SCH 23390 on the Cellular Content of Preproenkephalin A mRNA in Rat Striatum: A Quantitative Non-radioactive In Situ Hybridization Study.

The effects of acute i.p. administration of selective dopamine (DA) receptor antagonists on the expression of preproenkephalin A (PPE A) mRNA was investigated in the adult rat striatum. Animals were injected with either (a) a selective D1 receptor antagonist SCH 23390 (0.25 mg/kg), (b) a selective D2 receptor antagonist raclopride (5 mg/kg), or (c) SCH 23388 (0.25 mg/kg), the (S)-enantiomer of SCH 23390. Control naive animals did not receive an injection. At specific time points following drug administration (1, 3 or 9 h), rats were killed and striatal tissue processed for in situ hybridization with an alkaline phosphatase-labelled oligonucleotide probe complementary to a portion of the rat PPE A cDNA. Treatment of rats with SCH 23388 did not affect the content of PPE A mRNA expressed by striatal cells at any time point. However, 1 h after SCH 23390 administration, a significant decrease in striatal PPE A mRNA was detected, reflected by a decrease in the cellular content of mRNA. No significant changes in PPE A mRNA were detected in raclopride-treated sections at this time point. In contrast, both 3 and 9 h after an injection of raclopride a significant increase in the cellular content of PPE A mRNA was detected in the striatum. No change in the cellular content of mRNA was detected in SCH 23390-treated rats at these two latter time points. Throughout the striatum approximately 46% of neurons were found to express PPE A mRNA, with the highest percentage of cells (55%) being detected in the mid-caudal striatum. No significant differences in striatal DA content were detected with any drug treatment using HPLC electrochemical detection methods. These results demonstrate that acute administration of the DA D1 and D2 receptor antagonists has contrasting effects on the cellular content of PPE A mRNA in the adult rat striatum. These effects may reflect changes in the rate of mRNA transcription which may be mediated by cAMP.

An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.

We have developed a method of non-radioactive in situ hybridization histochemistry using alkaline phosphatase-labeled oligonucleotide probes to detect gene expression in the intestine. Because the intestine contains a large amount of endogenous alkaline phosphatase activity, mild acid pretreatment of the tissue sections was required to inactivate the alkaline phosphatase. Acid pre-treatment dramatically reduced the endogenous activity without affecting the efficiency of hybridization or the probe's ability to reveal a positive mRNA signal. Furthermore, the addition of polyvinyl alcohol to the substrate solution helped to keep the background staining low without adversely affecting the intensity of the signal. The current protocol allows rapid and sensitive detection of sites of gene expression in intestinal tissue.

Non-radioactive DNA probe for the rapid identification of Mycobacterium avium complex from clinical specimens

We evaluated an alkaline phosphatase labelled oligonucleotide probe (SNAP(TM), Syngene Co., Molecular Biosystems, Inc., San Diego CA) for the direct culture identification of Mycobacterium avium complex (MAC) isolated from clinical specimens. Mycobacterial species identified by conventional biochemical methods were retested with this DNA probe using the Centri-Dot(TM) format. The probe accurately identified all 69 pigmented M. avium complex and 15 non-pigmented isolates of M. avium complex. There were no false-positives with 45 non-MAC mycobacteria isolates (10 species) and 16 non-mycobacteria organisms (10 species). The sensitivity and specificity of the SNAP(TM) culture identification for M. avium complex were 100%. The alkaline phosphatase labelled DNA probe is stable for at least 9 months. The procedure can be completed within 2 h and is easily adapted in the clinical laboratory. For the strains encountered in our laboratory, we conclude that the SNAP(TM) hybridization is a rapid, specific, and reliable method for culture identification of M. avium complex.

Genotypic identification of pathogenic Mycobacterium species by using a nonradioactive oligonucleotide probe

Commercial DNA hybridization assays (Syngene, Inc., San Diego, Calif.) utilizing alkaline phosphatase-labeled oligonucleotide probes for the identification of Mycobacterium tuberculosis complex and M. avium complex (MAC) were evaluated with 261 isolates of mycobacteria. On the basis of biochemical criteria, the test for MAC was 98% specific and more sensitive (95 of 99, 95%) than Gen-Probe (88 of 99, 89% sensitivity); the major difference in sensitivity noted between the two systems was related to the hybridization of seven MAC strains to the SNAP X probe. The M. tuberculosis complex probe correctly identified all 62 isolates of M. tuberculosis and all 11 isolates of M. bovis, for a sensitivity of 100%. There were two discrepant reactions with mycobacteria other than M. tuberculosis complex isolates.

Improved method for the routine identification of toxigenic Escherichia coli by DNA amplification of a conserved region of the heat-labile toxin A subunit

This report describes a DNA amplification procedure for routine identification of heat-labile-toxin-producing Escherichia coli. Two oligonucleotide primers were used in a polymerase chain reaction procedure to amplify a highly conserved region of the A subunit of the heat-labile enterotoxin gene. Amplifications were done directly on E. coli colonies from plates when Salmonella, Shigella, or parasite infections were excluded as agents of the severe diarrhea in the patients. The conditions for the polymerase chain reaction method were empirically determined, and the procedure is inexpensive, sensitive, and specific. Positive results can be obtained over a wide variation in bacterial numbers, with no inhibition of Thermus aquaticus DNA polymerase. Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of this pathogen in clinical isolates. If greater sensitivity and specificity are required, hybridization with 32P- or alkaline phosphatase-labeled oligonucleotide probes can be used. Our results suggest that heat-labile-toxin-producing E. coli is responsible for about 9% of nondiagnosed diarrhea cases in Tygerberg Hospital, Tygerberg, Republic of South Africa.

Dopaminergic D 1 and D 2 receptor antagonists decrease prosomatostatin mRNA expression in rat striatum

The effect of acute dopamine receptor antagonist treatment on cellular prosomatostatin mRNA expression was investigated in the adult rat striatum using the technique of non-radioactive in situ hybridization. Adult female Wistar rats were given a single intraperitoneal injection of either raclopride (D 2 antagonist), SCH 23390 (D 1 antagonist) or the D 1 (S) enantiomer SCH 23388. Animals were killed either 1, 3 or 9 h following the single i.p. injection and their brains rapidly removed. Striatal sections were then processed for in situ hybridization using an alkaline phosphatase-labelled oligonucleotide probe complementary to a portion of the rat somatostatin cDNA. Blockade of dopamine D 1 and D 2 receptors resulted in a significant decrease in the cellular content of prosomatostatin mRNA. However, no change in the number of prosomatostatin mRNA containing striatal cells was observed following any of the treatments at any time point. These findings demonstrate that the cellular content of prosomatostatin mRNA in the adult rat striatum is influenced by selective dopamine D 1 and D 2 receptor antagonists. Further, these findings are consistent with a functional interaction between dopamine and somatostatin in the rat striatum.

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.

Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods.