scholarly journals Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis

2006 ◽  
Vol 55 (8) ◽  
pp. 1071-1077 ◽  
Author(s):  
Ranjana Srivastava ◽  
D. Kumar ◽  
M. N. Waskar ◽  
Meera Sharma ◽  
V. M. Katoch ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Hybridization ◽  
Repetitive Sequence ◽  
Repetitive Sequences ◽  
Subtractive Hybridization ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Nucleotide Sequencing ◽  
Family Gene ◽  
Positive Hybridization

A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a λgt11 library of M. tuberculosis by DNA–DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis.

scholarly journals Efficient anchoring of Erianthus arundinaceus chromatin introgressed into sugarcane by specific molecular markers

2020 ◽  
Author(s):  
Yongji Huang ◽  
Jiayun Wu ◽  
Xueting Li ◽  
Fan Yu ◽  
Xuguang Hu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
High Throughput ◽  
Repetitive Sequence ◽  
Repetitive Sequences ◽  
Distribution Patterns ◽  
Subtractive Hybridization ◽  
High Accuracy ◽  
Wild Relatives ◽  
45S Rdna ◽  
Erianthus Arundinaceus

Abstract Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking the inherited status of this chromosome in sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

scholarly journals Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission

2010 ◽  
Vol 76 (7) ◽  
pp. 2313-2325 ◽  
Author(s):  
Bijay K. Khajanchi ◽  
Amin A. Fadl ◽  
Mark A. Borchardt ◽  
Richard L. Berg ◽  
Amy J. Horneman ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Dna Hybridization ◽  
The United States ◽  
Clinical Isolates ◽  
Pulse Field ◽  
Clinical Samples ◽  
Aeromonas Species ◽  
Cytotoxic Activities ◽  
Molecular Fingerprinting ◽  
Suggestive Evidence

ABSTRACT A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

scholarly journals Efficient anchoring of Erianthus arundinaceus chromatin introgressed into sugarcane by specific molecular markers

2020 ◽  
Author(s):  
Yongji Huang ◽  
Jiayun Wu ◽  
Xueting Li ◽  
Fan Yu ◽  
Xuguang Hu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
High Throughput ◽  
Repetitive Sequence ◽  
Repetitive Sequences ◽  
Distribution Patterns ◽  
Subtractive Hybridization ◽  
High Accuracy ◽  
Wild Relatives ◽  
45S Rdna ◽  
Species Specific

Abstract Background: Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. Results: In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking the inherited status of this chromosome in sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. Conclusions: We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

scholarly journals In Vitro Activity of β-Lactams in Combination with β-Lactamase Inhibitors against Mycobacterium tuberculosis Clinical Isolates

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Fu Li ◽  
Li Wan ◽  
Tongyang Xiao ◽  
Haican Liu ◽  
Yi Jiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Synergetic Effect ◽  
Drug Susceptibility ◽  
Clinical Isolates ◽  
Nucleotide Sequencing ◽  
Drug Susceptibility Test ◽  
Single Target ◽  
Encoding Genes

Objectives. Evaluating the activity of nineteen β-lactams in combination with different β-lactamase inhibitors to determine the most potent combination against Mycobacterium tuberculosis (MTB) in vitro. Methods. Drug activity was examined by drug susceptibility test with 122 clinical isolates from China. Mutations of blaC and drug targets ldtMt1, ldtMt2, dacB2, and crfA were analyzed by nucleotide sequencing. Results. Tebipenem (TBM) in combination with clavulanate (CLA) exhibited the highest anti-TB activity. The MIC of β-lactam antibiotics was reduced most evidently in the presence of CLA, compared to avibactam (AVI) and sulbactam (SUB). Eight polymorphism sites were identified in blaC, which were not associated with β-lactams resistance. Interestingly, one strain carrying G514A mutation in blaC was highly susceptible to β-lactams regardless of the presence of inhibitors. The transpeptidase encoding genes, ldtMt1, ldtMt2, and dacB2, harboured three mutations, two mutations, and one mutation, respectively, but no correlation was found between these mutations and drug resistance. Conclusion. The activity of β-lactams against MTB and different synergetic effect of β-lactamase inhibitors were indicated. TBM/CLA exhibited the most activity and has a great prospect in developing novel anti-TB regimen; however, further clinical research is warranted. Moreover, the resistance to the β-lactam antibiotics might not be conferred by single target mutation in MTB and requires further studies.

scholarly journals Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Wansadaj Jaroenram ◽  
Jantana Kampeera ◽  
Narong Arunrut ◽  
Sarawut Sirithammajak ◽  
Sarinya Jaitrong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Hybridization ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Resource ◽  
Lateral Flow Dipstick ◽  
Probe System ◽  
One Step

Abstract Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.

scholarly journals Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of gyrA Mutations in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates

2004 ◽  
Vol 48 (2) ◽  
pp. 596-601 ◽  
Author(s):  
Augustine F. B. Cheng ◽  
Wing W. Yew ◽  
Edward W. C. Chan ◽  
Miu L. Chin ◽  
Mamie M. M. Hui ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multiplex Pcr ◽  
Region Of Interest ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Nucleotide Sequencing ◽  
Phenotypic Resistance ◽  
New Strategy ◽  
High Level

ABSTRACT A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains ≤ 4 mg/liter) were apparently more active than ofloxacin, levofloxacin, and sparfloxacin (MIC for 90% of strains ≥ 16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains ≤ 1 mg/liter). Another 83 isolates without mutations were fully susceptible to all six fluoroquinolones (ofloxacin MIC for 90% of strains = 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoroquinolones among M. tuberculosis clinical isolates, which appears to be predominantly due to gyrA mutations, may be readily detected by genotyping techniques such as multiplex PCR amplimer conformation.

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

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