scholarly journals Evaluation ofhsp65Nested PCR-Restriction Analysis (PRA) for Diagnosing Tuberculosis in a High Burden Country

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Sara Macente ◽  
Clarice Queico Fujimura Leite ◽  
Adolfo Carlos Barreto Santos ◽  
Vera Lúcia Dias Siqueira ◽  
Luzia Neri Cosmo Machado ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Restriction Analysis ◽  
Mycobacterial Infection ◽  
Acid Fast Bacillus ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Specific Diagnosis ◽  
Acid Fast Bacillus Smear ◽  
Selection Of

Current study evaluated thehsp65Nested PCR Restriction Fragment Length Polymorphism Analysis (hsp65Nested PCR-PRA) to detect and identifyMycobacterium tuberculosiscomplex directly in clinical samples for a rapid and specific diagnosis of tuberculosis (TB).hsp65Nested PCR-PRA was applied directly to 218 clinical samples obtained from 127 patients suspected of TB or another mycobacterial infection from July 2009 to July 2010. Thehsp65Nested PCR-PRA showed 100% sensitivity and 95.0 and 93.1% specificity in comparison with culture and microscopy (acid fast bacillus smear), respectively.hsp65Nested PCR-PRA was shown to be a fast and reliable assay for diagnosing TB, which may contribute towards a fast diagnosis that could help the selection of appropriate chemotherapeutic and early epidemiological management of the cases which are of paramount importance in a high TB burden country.

scholarly journals Identification of Bartonella Species Directly in Clinical Specimens by PCR-Restriction Fragment Length Polymorphism Analysis of a 16S rRNA Gene Fragment

1999 ◽  
Vol 37 (12) ◽  
pp. 4045-4047 ◽  
Author(s):  
Ghassan M. Matar ◽  
Jane E. Koehler ◽  
Georgia Malcolm ◽  
Mary Ann Lambert-Fair ◽  
Jordan Tappero ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Restriction Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Clinical Specimens ◽  
Restriction Fragment Length

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, andB. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis usingDdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintanaamplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethaegave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique “signature” restriction patterns characteristic for each species were obtained. These patterns were useful in identifying theBartonella species associated with each tissue specimen.

scholarly journals Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

scholarly journals Nested PCR and New Primers for Analysis of Sulfate-Reducing Bacteria in Low-Cell-Biomass Environments

2010 ◽  
Vol 76 (9) ◽  
pp. 2856-2865 ◽  
Author(s):  
Ludovic Giloteaux ◽  
Marisol Goñi-Urriza ◽  
Robert Duran
Keyword(s):  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Polymorphism Analysis ◽  
Direct Pcr ◽  
Sulfate Reducing ◽  
Cell Biomass ◽  
Low Levels ◽  
Reducing Bacteria ◽  
First Time

ABSTRACT New primers were designed for the amplification of dsrAB genes by nested PCR to investigate the diversity of sulfate-reducing prokaryotes (SRP) in environments with low bacterial cell density. The success of the nested PCR for the determination of SRP diversity was estimated by terminal-restriction fragment length polymorphism analysis in the Reigous, a small creek at an inactive mine (Carnoulès, France), which constitutes an extreme acidic arsenic-rich environment. Nested PCR limits were evaluated in dsrAB-rich sediments, and this technique was compared to direct PCR using either known primers (DSR1F/DSR4R) or new primers (dsr619AF/dsr1905BR). The comparison of clone libraries revealed that, even if the levels of diversity observed were not identical, nested PCR did not reduce the diversity compared to that of direct DSR1F/DSR4R PCR. Clone sequences were affiliated mainly with the Desulfobacteraceae and Desulfohalobiaceae families. Many sequences (∼30%) were related to a deeply branching lineage unaffiliated with any cultured SRP. Although this dsrAB cluster was found in all libraries, the new primers better amplified this lineage, providing more information on this unknown bacterial group. Thanks to these new primers in nested PCR, the SRP community from Carnoulès could be characterized. Specific SRP populations were obtained according to environmental characteristics. Desulfomicrobiaceae-related sequences were recovered in samples with low pH, low levels of dissolved oxygen, and high As content, while sequences belonging to the deeply branching group were found in a less extreme sample. Furthermore, for the first time, dsrAB sequences related to the latter group were recovered from freshwater.

scholarly journals Molecular Characterization of Korean Bacillus anthracis Isolates by Amplified Fragment Length Polymorphism Analysis and Multilocus Variable-Number Tandem Repeat Analysis

2005 ◽  
Vol 71 (8) ◽  
pp. 4664-4671 ◽  
Author(s):  
Chunsun Ryu ◽  
Kyunghee Lee ◽  
Han-Jun Hawng ◽  
Cheon-Kwon Yoo ◽  
Won-Keun Seong ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Length Polymorphism ◽  
Tandem Repeat ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Variable Number Tandem Repeat ◽  
Amplified Fragment Length Polymorphism ◽  
Variable Number ◽  
Clinical Samples ◽  
Polymorphism Analysis

ABSTRACT We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.

scholarly journals Q Fever in the Greek Island of Crete: Detection, Isolation, and Molecular Identification of Eight Strains of Coxiella burnetii from Clinical Samples

1998 ◽  
Vol 36 (7) ◽  
pp. 2063-2067 ◽  
Author(s):  
Ioanna Spyridaki ◽  
Achilleas Gikas ◽  
Diamantis Kofteridis ◽  
Anna Psaroulaki ◽  
Yannis Tselentis
Keyword(s):  
Coxiella Burnetii ◽  
Nested Pcr ◽  
Q Fever ◽  
Buffy Coat ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Antibody Technique ◽  
Presenting Symptoms ◽  
Specificity And Sensitivity ◽  
Reference Strains

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the Coxiella burnetii strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of Coxiella burnetii in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of Coxiella burnetii infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).

scholarly journals First Report in Mauritius of Bacterial Leaf Blight of Syngonium Caused by Xanthomonas campestris pv. syngonii

Plant Disease ◽  
2008 ◽  
Vol 92 (6) ◽  
pp. 980-980 ◽  
Author(s):  
E. Jouen ◽  
I. Robène-Soustrade ◽  
L. Gagnevin ◽  
O. Pruvost ◽  
S. Benimadhu
Keyword(s):  
United States ◽  
Nested Pcr ◽  
Xanthomonas Campestris ◽  
Restriction Analysis ◽  
The United States ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Polymorphism Analysis ◽  
Pcr Assay ◽  
Buffer Solution

In November of 2006, necrotic leaf lesions with water-soaked margins were observed on Syngonium podophyllum in Floréal, Forest Side, and Réduit, Mauritius. Although not an economically important crop, the disease was of concern because syngonium is a host for Xanthomonas axonopodis pv. dieffenbachiae and the anthurium industry is of major economic importance in Mauritius. X. campestris pv. syngonii, described as the causal agent of bacterial leaf blight of syngonium (2), is genetically closely related to group 9.4 X. axonopodis pv. dieffenbachiae strains (3). In contrast to X. axonopodis pv. dieffenbachiae, X. campestris pv. syngonii strains are highly virulent on syngonium but are not pathogenic on anthurium or other Araceae, but both react similarly to the Xcd108 monoclonal antibody (Mab) (Agdia Inc., Elkhart, IN) and to a nested PCR assay designed for X. axonopodis pv. dieffenbachiae (4). X. axonopodis pv. dieffenbachiae and X. campestris pv. syngonii strains can be distinguished on the basis of restriction analysis of the amplicon of this PCR assay. Four pure cultures isolated from S. podophyllum were gram negative, yellow pigmented, and produced mucoid colonies on yeast peptone glucose agar (YPGA). One positive control strain of X. campestris pv. syngonii (LMG 9055 from the United States) and X. axonopodis pv. dieffenbachiae (LMG 695 from Brazil) were also used for all tests. All strains reacted positively with the Xcd108 MAb using indirect ELISA. DNA from all strains was amplified by the nested PCR assay, and the HincII restriction pattern of the amplicons identified strains from Mauritius as X. campestris pv. syngonii. Pathogenicity tests were performed on 8-month-old plants of Anthurium andreanum cv. Florida, Dieffenbachia maculata cv. Tropic Marianne, and S. podophyllum cv. Robusta by infiltrating suspensions containing ~1 × 105 CFU ml¯1 of each strain prepared from YPGA plates. Each strain was inoculated onto three young leaves (four inoculation sites per leaf) on two plants. Negative control plants received sterile Tris buffer solution (10 mM, pH 7.2). Plants were maintained in a growth chamber with day and night temperatures at 30 ± 1°C and 26 ± 1°C, respectively, 95 ± 5% relative humidity, 30 μmol·m¯2·s¯1 light intensity, and a photoperiod of 12 h (4). All strains caused typical water-soaked lesions 14 days after inoculation (dai) on syngonium. Lesions turned necrotic with chlorotic margins 27 to 34 dai. Typical bacterial blight lesions were observed on anthurium leaves inoculated with X. axonopodis pv. dieffenbachiae strain LMG 695, but no symptoms were observed 60 dai when strains from Mauritius and LMG 9055 were used. Amplified fragment length polymorphism analysis of four strains from Mauritius and additional reference, X. axonopodis pv. dieffenbachiae and X. campestris pv. syngonii strains, using SacI/MspI and four primer pairs (unlabeled MspI+1 [A, C, T, or G] primers and 5′-labeled-SacI+C primer for the selective amplification step) (1), showed that the strains from Mauritius could be distinguished from X. axonopodis pv. dieffenbachiae but were identical to X. campestris pv. syngonii strains from the United States and Réunion Island. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) R. S. Dickey and C. H. Zumoff. Phytopathology 77:1257, 1987. (3) J. L. W. Rademaker et al. Phytopathology 95:1098, 2005. (4) I. Robene-Soustrade et al. Appl. Environ. Microbiol. 72:1072, 2006.

scholarly journals Nonculture Prediction of Neisseria meningitidis Susceptibility to Penicillin

2001 ◽  
Vol 45 (12) ◽  
pp. 3625-3628 ◽  
Author(s):  
Aude Antignac ◽  
Jean-Michel Alonso ◽  
Muhamed-Kheir Taha
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Neisseria Meningitidis ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Penicillin G ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Restriction Fragment Length

ABSTRACT We developed a nonculture method to predict the susceptibility ofNeisseria meningitidis to penicillin G. ThepenA gene was amplified and submitted to restriction fragment length polymorphism analysis. This approach was first validated with a collection of 75 meningococcal strains of known phenotypes. It was next successfully applied to 29 clinical samples.

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