Application on in situ PCR methods in histochemistry

1995 ◽  
Vol 53 ◽  
pp. 754-755
Author(s):  
Omar Bagasra
Keyword(s):  
Thermostable Enzyme ◽  
Small Sample ◽  
Marker Genes ◽  
Infected Cell Line ◽  
Gene Sequences ◽  
Minute Amount ◽  
In Situ Pcr ◽  
Pcr Method ◽  
Polymerase Chain

Since the publication of the first report regarding the in situ amplification of HIV-1 gag gene in a HIV-l infected cell line in 1990, there has been an explosion of research in the area of in situ PCR. There are over 200 publications describing various forms of in situ gene, identifying various infectious agents, tumor marker genes and other genetic elements of interest, in peer reviewed journals (reviewed in 2-15). The polymerase chain reaction (PCR) method for amplification of defined gene sequences has proved a valuable tool not only for basic researchers but also for clinical scientists. Using even a minute amount of DNA or RNA and choosing a thermostable enzyme from a large variety of sources, one can enlarge the amount of the gene of interest, which can be analyzed and/or sequenced. Thus genes or portions of gene sequences present only in a small sample of cells or small fraction of mixed cellular populations can be examined.

scholarly journals Generation of a whole chromosome painting probe from monochromosomal hybrid cells by the alu-polymerase chain reaction

2007 ◽  
Vol 59 (2) ◽  
pp. 89-95 ◽  
Author(s):  
Danijela Drakulic ◽  
Gordana Nikcevic ◽  
Vesna Djordjevic ◽  
Milena Stevanovic
Keyword(s):  
Chromosome Painting ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Rearrangements ◽  
Painting Probe ◽  
Complex Chromosomal Rearrangements ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Complex Chromosome ◽  
Whole Chromosome Painting

Fluorescent in situ hybridization (FISH) has become a widespread technique applicable in basic science and diagnostics. Chromosome painting represents a special application of FISH that has found increasing use in identification of complex chromosome rearrangements. Here we present a version of the Alu-PCR method modified to generate a whole chromosome painting probe (WCP) for human chromosome 19 using monochromosomal cell hybrids. In setting up conditions for this method, we established a cheap and fast approach to generation of WCPs for other human chromosomes that could be particularly useful for unambiguous identification of complex chromosomal rearrangements associated with cancer. .

scholarly journals Detection ofMycobacterium aviumsubsp.paratuberculosisby a Direct In Situ PCR Method

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Fernando Delgado ◽  
Diana Aguilar ◽  
Sergio Garbaccio ◽  
Gladys Francinelli ◽  
R. Hernández-Pando ◽  
...  
Keyword(s):  
Negative Control ◽  
Tissue Samples ◽  
In Situ Pcr ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Pcr Method ◽  
In Situ Detection ◽  
Formalin Fixed ◽  
Healthy Calf

In situ detection ofMycobacterium aviumsubsp.paratuberculosisis useful for diagnosis and research of paratuberculosis. The aim of this paper was to detect this agent in formalin-fixed, paraffin-embedded tissue samples by a direct in situ PCR. The technique was performed on ileum or ileocaecal lymph node samples from 8 naturally infected cattle and 1 healthy calf, by using p89 and p92 primers for amplification of IS900 sequence. Moderate positive signal was detected in all positive samples and not in negative control, but tissues resulted were affected in many cases due to the enzymatic treatment and the high temperature exposition. Although the technique was useful for Map detection, the signal was lower than immunohistochemistry probably because of the fixation process. In one case, signal was higher, which might be due to the detection of spheroplasts. Thus, the described method should be recommended when others resulted negative or for spheroplasts detection.

Enterovirus is Not Present in Placentas from Cases of Perinatal Depression Using Polymerase Chain Reaction Analysis

2009 ◽  
Vol 12 (3) ◽  
pp. 177-179 ◽  
Author(s):  
Jesse Cover ◽  
Edwina Popek ◽  
Myra Wyckoff ◽  
N. Kristine Leos ◽  
Beverly Barton Rogers
Keyword(s):  
Polymerase Chain Reaction ◽  
Respiratory Insufficiency ◽  
Pcr Amplification ◽  
Polymerase Chain Reaction Analysis ◽  
Chain Reaction ◽  
Apgar Scores ◽  
Specific Patient ◽  
In Situ Pcr ◽  
Polymerase Chain

Enteroviruses have been implicated as a cause of low Apgar scores in conjunction with perinatal seizures and respiratory insufficiency. Using in-situ reverse transcriptase polymerase chain reaction (in-situ PCR), Nuovo et al detected enterovirus in up to 86% of placentas from perinates exhibiting these symptoms. In-situ PCR has been the only method employed to assess for the presence of enterovirus in this specific patient population. The purpose of our study was to use PCR amplification of enterovirus from extracted RNA to confirm these observations. RNA was extracted from 26 placentas of infants with low Apgar scores, perinatal seizures, and respiratory insufficiency. Each extraction was positive for beta-actin RNA, which confirmed that the integrity of RNA was maintained in the sample. Enterovirus RNA was not detected in any of the cases. Our results indicate that enterovirus is not present in placentas from neonates with the combination of low Apgar scores, respiratory insufficiency, and seizures, as previously reported.

scholarly journals Identification of Eutypa lata by PCR-RFLP

Plant Disease ◽  
2004 ◽  
Vol 88 (9) ◽  
pp. 925-929 ◽  
Author(s):  
P. E. Rolshausen ◽  
F. Trouillas ◽  
W. D. Gubler
Keyword(s):  
Universal Primers ◽  
Phaeomoniella Chlamydospora ◽  
Phomopsis Viticola ◽  
Eutypa Lata ◽  
Incorrect Diagnosis ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Rflp Assay

Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.

scholarly journals Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4–Anaplastic Lymphoma Kinase Fusion–Positive Non–Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing

2012 ◽  
Vol 136 (7) ◽  
pp. 796-803 ◽  
Author(s):  
Michelle L. Wallander ◽  
Katherine B. Geiersbach ◽  
Sheryl R. Tripp ◽  
Lester J. Layfield
Keyword(s):  
Polymerase Chain Reaction ◽  
Small Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Small Cell Lung ◽  
Chain Reaction ◽  
Anaplastic Lymphoma ◽  
Pcr Method ◽  
Polymerase Chain

Context.—Echinoderm microtubule–associated proteinlike 4–anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non–small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection. Objective.—To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions. Design.—Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n  =  42; mutant, n  =  4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b). Results.—EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation. Conclusions.—The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

scholarly journals Comparison of Three Strategies for Sequencing Rabies Viral G Gene

2013 ◽  
Vol 2 (1) ◽  
Author(s):  
Shengli Meng ◽  
Wenli Lv ◽  
Jie Wu ◽  
Jilin Wang ◽  
Gelin Xu ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Low Cost ◽  
Cost Effective ◽  
Brain Homogenate ◽  
Universal Primers ◽  
Viral Gene ◽  
Gene Sequences ◽  
Rt Pcr ◽  
Pcr Method ◽  
Polymerase Chain

<p>It is essential to rapidly, low-cost and precisely determining gene sequences of rabies virus. Reverse transcription-polymerase chain reaction can be used to identify rabies viral G gene sequences. In this study, we evaluated three methods, conventional RT-PCR and direct sequencing, adapter RT-PCR and sequencing with universal primers, conventional RT-PCR and cloning sequencing with universal primers, to detect rabies in animal brain homogenate. Four rabies isolates recovered from Fuyang city of Anhui province were diagnosed as positive using the fluorescentantibody test, rapid rabies enzyme immunodiagnosis methods and the mouse inoculation test. The results indicated that the adapter RT-PCR method and sequencing with universal primers is extremely well suited for sequencing rabies viral gene, which is rapid, cost-effective and precise compared with other two methods.<strong></strong></p>

scholarly journals DETECTION AND SEQUENCING GENES IRON, IUTA, AND HLYF IN AVIAN PATHOGENIC ESCHERICIA COLI

2019 ◽  
pp. 229
Author(s):  
Nyoman Anandiya Ramaditya ◽  
I Nengah Kerta Besung ◽  
I Gusti Ngurah Kade Mahardika
Keyword(s):  
The Other ◽  
Marker Genes ◽  
Chain Reaction ◽  
Gene Markers ◽  
E Coli ◽  
The World ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Escherichia Coli Bacteria ◽  
Pathogenic Gene

Research has been carried out to detect pathogenic gene markers of Avian Pathogenic Eschericia coli (APEC) iroN, iutA, and hlyF in Escherichia coli bacteria isolated from organs of sick chickens in Bali, and to determine phylogenetic relationships between those marker genes in Bali and in the other countries in the world. Six isolates of E. coli bacteria with codes E2, E3, E7, E8, E9, and E10 were used in this study. The isolates were isolated from domestic chicken in 2018. All genes were detected using the Polymerase Chain Reaction (PCR) method. The genes of iroN, iutA, and hlyF could be detected from all isolates. Well readable sequence of iroN, hlyF, and iutA was 659 bp, 518 bp, and 250 bp, respectively. All three genes were homogenous. Phylogical analysis shows that all pathogenic markers share same cluster with the pathogenic E. coli from all countries in the world.

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