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Search Results: 1 - 10 of 9618 matches for " Multiplex PCR "
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"PCR Application In Reognition Of Prevelant Deletion Of α Globin Gene In Alpha Thalasemia Carriers "
R. Kiani-Shirazi,S. Zainali,M. Karimipoor,B. Zarbakhsh R. Alibakhshi
Tehran University Medical Journal , 2006,
Abstract: Background and Aim: -thalassemia is the most common inherited disorder of hemoglobin (Hb) synthesis in the world. Alpha thalassemia most frequently results from the loss of one (- ) or both (- -) of the duplicated genes ( ) on chromosome 16. Carriers of deletional forms of -thalassemia (- / - /- , or --/ ) are clinically normal but have a mild hypochromic, microcytic anemia. Compound heterozygotes (--/- ) called Hb H disease. Fetuses who inherit no genes (--/--) (Hb Bart's Hydrops fetalis syndrome) die either inutero or shortly after birth, More than 95% of recognized -thalassemia involves deletion of one or both globin genes on chromosome 16. Materials and Methods: The assay was tested on 114 Iranian individuals with low MCV and MCH levels but normal HbA2 who had not responded to Iron treatment. patients was referred to the Department of Biotechnology, Pasteur Institute of Iran by Health Centers. Genomic DNA was isolated from white blood cells by salting out method. We have developed a reliable, single - tube multiplex polymerase chain reaction (PCR) assay for the 7 most frequent - thalassemia deletions (-- SEA , --THAI, --FIL , -α20.5 , --MED, -α4.2 , -α3.7). Results: DNA fromd thalassemia carriers was tested for the presence of different types of globin gene deletion (s). The - 3.7 and - 4.2 single gene deletions, and the Mediterranean (-- MED and - 20.5) double gene deletions were found in some samples. Conclusion: The - 3.7 deletion was found to be the most common cause of globin gene deletion in our samples. Multiplex PCR for α gene deletion analysis is simple, rapid and sensitive.
Identification of sterile cytoplasm (CMS) in maize by using specific mtDNA primers
Ignjatovi?-Mici? Dragana,Nikoli? Ana,Mladenovi?-Drini? Sne?ana,Van?etovi? Jelena
Genetika , 2006, DOI: 10.2298/gensr0603227i
Abstract: Thirty sources of cytoplasmic male sterility (CMS) from Maize Gene Bank "Zemun Polje", distributed among Yugoslav OP varieties, have been tested for the presence of particular type of cytoplasm by a single seed multiplex PCR approach with specific primer pairs for T, C and S type cytoplasm. Combination of three pairs of primers in a single PCR reaction, corresponding to the chimeric regions of mtDNA sequences specific for each type of CMS, allowed reliable identification of the major CMS types. Dominant presence of S type cytoplasm was detected. For sources where there is no clear identification of the type of CMS (absence of the PCR band) there is a reasonable doubt that it could be a new, yet unidentified type of CMS.
Multiplex PCR on Leptospiral Isolates from Kolenchery, Kerala, India
Sugathan S,Varghese T
Indian Journal of Medical Microbiology , 2005,
Abstract: Human leptospirosis causes severe multiorgan dysfunctions that may end in multiorgan failure and death. The methods in hand for diagnosis of leptospirosis like culture, ELISA and MAT only help to confirm the disease, and are of little value in early detection. The aim of this study was to find out if the two sets of primers described earlier could detect all the isolates from the area, for the purpose of using the resultant database for early detection of leptospires in future from clinical specimens. The study was done on culture isolates from Jan 2000 to June 2002 attending the department of medicine, MOSC medical college hospital, Kolenchery, Kerala, India. DNA of 45 culture isolates were amplified by multiplex PCR using two sets of previously described primers, G1, G2 and B 64-I, B 64-II. Specific amplifications of either 285 or 563 bp size were obtained from all isolates included in the study indicating the utility of the multiplex PCR in the rapid detection of leptospires in clinical samples.
Development and Evaluation of a Multiplex PCR for the Detection of Campylobacter concisus and Other Campylobacter spp. from Gastroenteritis Cases  [PDF]
Mohsina Huq, Gena Gonis, Taghrid Istivan
Open Journal of Medical Microbiology (OJMM) , 2014, DOI: 10.4236/ojmm.2014.41005
Abstract:
We developed and evaluated a multiplex PCR (m-PCR) for application in routine diagnostic laboratories to detect Campylobacter spp. in stool samples including C. concisus, C. jejuni, and C. coli. When this m-PCR was applied on spiked faecal samples, C. concisus, C. jejuni, and C. coli were specifically identified at 105 cells/gm of faeces. To compare the sensitivity of the m-PCR with conventional culture techniques, the same spiked stool samples were cultured on an antibiotic free Columbia blood agar using the filtration technique. The detection limit of conventional culture method was 105 cells/gm of stool for C. concisus and 106 cells/gm of stool for C. jejuni and C. coli. The m-PCR was applied to test 127 faecal samples from children with gastroenteritis and the results were compared with the conventional bacterial cultures data. By this m-PCR technique, C. jejuni was detected in 7 samples, C. coli in 2 samples, and C. concisus in 7 samples. However, the conventional culture results for these samples were 6 for C. jejuni, 2 for C. coli and only one sample was positive for C. concisus. In total, 19 samples were positive for Campylobacter spp. by m-PCR while only 9 samples were positive for Campylobacter spp. by culture. In conclusion, m-PCR is more sensitive than the culture technique to detect C. concisus and other fastidious campylobacters in faeces.
Two-Tube Multiplex PCR for Genotyping Tuberculous and Nontuberculous Mycobacterial Species in Pathology Specimens  [PDF]
Michael Dictor, Janina Warenholt, Marta Lukasiewicz
Advances in Microbiology (AiM) , 2018, DOI: 10.4236/aim.2018.81003
Abstract: Background: The incidence of mycobacterial infection, in particular M. tuberculosis complex (MTC), is increasing in some Western countries, while nontuberculous mycobacteria (NTM) may be recognized more frequently in clinical specimens worldwide. The clinical scenario and available histopathology alone are often insufficient to separate these two categories of mycobacterial disease, whose behavior and treatment differ. In particular, NTM may be clinically unsuspected in pathological specimens and the opportunity for culturing missed. Methods: We developed two multiplex PCR assays, which distinguish MTC from NTM by detecting the IS6110 insert in the first tube and discriminating up to 14 NTM reference strains in the second by targeting the 16S-23S rRNA internal transcribed spacer. Test material included 594 routine clinical specimens with diverse pathology; many were granulomas unrelated to mycobacterial infection. About 75% were formalin-fixed paraffin blocks, the remainder mainly cytologic imprints or aspirates on FTA cards submitted on suspicion of mycobacterial infection either to avoid frozen sectioning (with the attendant risk of aerosolisation) or at the time of fine needle aspiration. Results: The paraffinized material yielded 53 MTC positives and the cytological 21 positives. A subset consisting of 337 specimens was also analyzed for NTM and yielded 51 positives. The frequency of simultaneous NTM infection in tuberculous patients was about 17%. Mycobacterium avium complex represented the dominant NTM species overall, showed a predilection for lung and lymph node, and together with M. haemophilum were the second most frequent NTM just behind M. ulcerans/M. marinum in skin and soft tissue, the category displaying the largest NTM diversity. Conclusions: Cytological and deparaffinized tissue analyzed in a new two-tube multiplex PCR allows for specific discrimination of causative agents in mycobacterial infection. MTC is readily distinguished from NTM for appropriate therapy, and NTM presumptively diagnosed at the species level allows appropriate choices of antimicrobials.
Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products
Pelisser, Marcia Regina;Klein, Cátia Silene;Ascoli, Kelen Regina;Zotti, Thaís Regina;Arisi, Ana Carolina Maisonnave;
Brazilian Journal of Microbiology , 2009, DOI: 10.1590/S1517-83822009000100025
Abstract: multiplex pcr was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and fema gene (specific for staphylococcus aureus) in coagulase-positive staphylococci (cps) isolated from cheese and meat products. from 102 cps isolates, 91 were positive for fema, 10 for sea, 12 for sed and four for see.
Comparison of Multiplex PCR-ELISA and conventional Multiplex PCR for detection of HIV-1/HCV co-infection
Mehrdad Ravanshad,Z Khanlari,M Rasouli,M Ziyaeyan
Iranian Journal of Microbiology , 2009,
Abstract: Background and Objectives: PCR is a sensitive assay and could be used as an accurate diagnostic method for detecting various types of microorganisms' genome in low concentration in biological specimens. The demand for sensitive, rapid, safe and easy detection of PCR products has led researchers to a combination of this method with ELISA."nMaterials and Methods: Conserved sequences were selected for design of primers. Samples were tested by ELISA (for detection of specific HIV and HCV proteins) and real- time PCR for detection of specific nucleic acid and viral genome respectively. Viral genome was extracted and reverse transcription was performed with M-Mulv and the cDNA kept at -80o C. The PCR products were labeled by DIG-dUTP. Diluted PCR products were analyzed with both electrophoresis and ELISA methods."nResults: Thirty-five samples were tested with the PCR-ELISA method. False positive or negative reactions were not observed. ELISA results of diluted products were compared with results obtained by electrophoresis. In gel electrophoresis, dilution of 1/10 was positive, but in ELISA, optical density of 1/100 dilution was much more than the cut-off value."nConclusion: Detection limits for gel electrophoresis as well as ELISA have been evaluated. It was shown that the PCR-ELISA method is ten times more sensitive than conventional PCR.
Multiplex PCR for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolated from Bandung, Indonesia
HERA NOVIANA,ZEILY NURACHMAN,MAELITA RAMDANI,AS NOER
Microbiology Indonesia , 2007,
Abstract: Mycobacterium tuberculosis resistant to rifampin and isoniazid, known as multidrug-resistant M. tuberculosis (MDR-TB) strains, is an emerging problem of great importance to public health, with higher mortality rates than for drug-sensitive strains. Rifampin resistance is due to mutations on the hot spot region of the rpoB gene, especially at positions 526 and 531, and isoniazid resistance is due to mutation on katG at position 315. Mechanisms of resistance are an appropriate target for molecular genotyping diagnostic methods. Here we examined the multiplex PCR assays for the rapid detection targeting rpoB526, rpoB531, and katG mutations. Sixty-one M. tuberculosis strains were studied based on rpoB526, rpoB531, and katG315 assays employing multiplex PCR. Of the 61 strains, the susceptibility tests determined 42 isolates were MDR-TB strains, 10, 4, and 5 isolates were resistant to rifampin, isoniazid, and at least to six drugs which prescibed for TB, respectively. The mutation profiles of the 42 MDR strains assayed by multiplex PCR were 81 and 38.1% on rpoB and katG, respectively. Six rifampin-resistant isolates (60%) had a mutation on rpoB, 25% isoniazid-resistant isolates had mutation on katG, and 20% of the isolates that were sensitive to all drugs tested had a mutation on rpoB. Sequencing analysis revealed sensitivity of the multiplex PCR assay for rpoB was 98.4% and was 100% for katG. There was a 19% difference between phenotype and genotype properties of all isolates detected. In conclusion, the sensitivity of multiplex PCR method was sufficient for preliminary detection of rpoB and katG mutations, but resistance M. tuberculosis to rifampin and isoniazid were not always conferred by mutated alleles on rpoB and, especially, on katG.
Development of two multiplex PCRs for microsatellite analysis in Alpine chamois (Rupicapra r. rupicapra)
D. Soglia,S. Sartore,S. Maione,F. La Neve
Italian Journal of Animal Science , 2010, DOI: 10.4081/ijas.2005.2s.61
Abstract: The study of the genetic diversity gives important information about structure, subdivision in subunits and evolution of populations. Chamois (Rupicapra rupicapra, Linneus 1758) are mountain ungulates belonging to the subfamily Caprinae. They are presently distributed over most of the medium to high altitude mountains in the Southern Europe. Ten distinct geographical populations have been recognised as subspecies (Masini and Lovari, 1988); one of this subspecies, R. r. rupicapra, includes also the chamois living on the Italian Alps.
Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR
Z Safari,M Saadati,Sh Nazarian,M Heiat
Journal of Shahid Sadoughi University of Medical Sciences , 2010,
Abstract: Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.
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