Routine Use of PCR–Reverse Cross-Blot Hybridization Assay for Rapid Identification of
            <i>Mycobacterium</i>
            Species Growing in Liquid Media

Sanguinetti, Maurizio; Posteraro, Brunella; Ardito, Fausta; Zanetti, Stefania Anna Lucia; Cingolani, A; Sechi, Leonardo Antonio; Luca, Andrea De; Ortona, L; Fadda, Giovanni

doi:10.1128/jcm.36.6.1530-1533.1998

Cited by 31 publications

(15 citation statements)

References 31 publications

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“…tested. The results showed that the sensitivity of PCR-RLB was significantly higher (about 100 times) than that of PCR, which is consistent with literature reports [23][24][25][26][27][28][29].…”

Section: Simultaneous Detection Of 7 Fly-borne Bacterial Pathogenic Msupporting

confidence: 91%

Simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms by the reverse line blot hybridization assay

Luo

Gou

et al. 2020

Preprint

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Background: As a widespread health pest, flies can carry more than 100 kinds of path ogenic microbes to threat human health, resulting in a wide range of disease infection and transmission. The aim of this study was to develop a sensitive, reliable and rapid method for the simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms, in order to effectively prevent and control fly-borne bacterial diseases. Results: PCR-RLB method could directly and accurately detect fly-borne bacteria species corresponding of 7 species-specific probes. The sensitivity of PCR-RLB was significantly higher (about 100 times) than that of PCR. At the same time, the membrane binding oligonucleotide species-specific probes prepared in RLB detection technology can be reused for detection of bacteria after washing with 0.5 M EDTA, which greatly improves the detection efficiency. This method was used to detect the intestinal pathogens carried by flies in four different areas of Lanzhou city. In 106 groups of samples from different areas, The numbers of carrying seven bacterial strains were 2 (S. aureus), 52 (S. flexneri), 0 (A. caviae), 3% (V. vulnificus), 56 (S. enterica), 1 (P. vulgaris) and 33 (Y. enterocolitica), respectively. Their proportions of 7 bacterial strains carried by houseflies were 1.23% ( S. aureus ), 32.1% ( S. flexneri ), 0% ( A. caviae ), 1.85% ( V. vulnificus ), 34.57% ( S. enterica ), 0.62% ( P. vulgaris ) and 20.37% ( Y. enterocolitica ), respectively. The results showed that there was a high correlation between the bacteria carrying rate of flies and the health environment in urban areas. S. enterica , S. flexneri and Y. enterocolitica accounted for the overwhelming majority of the seven pathogenic strains carried by houseflies. This indicated that houseflies played an important role in the transmission of intestinal infectious diseases. S. aureus and V. vulnificus were carried by houseflies near the hospital area indicates that hospitals should do well in killing and controlling flies and further strengthen the prevention and control of fly-borne bacterial diseases. Conclusion: The developed PCR-RLB assay have potential clinical application in the simultaneous detection of fly-borne bacterial species.

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Section: Simultaneous Detection Of 7 Fly-borne Bacterial Pathogenic Msupporting

confidence: 91%

Simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms by the reverse line blot hybridization assay

Luo

Gou

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…In oligonucleotide-speci¢c capture plate hybridization (OSCPH) the digoxigenin-labeled amplicon is hybridized with a biotinylated M. genavense-speci¢c oligonucleotide and captured on a streptavidin-coated plate [19]. Ampli¢cation with biotinylated primers and analysis of the PCR products by reverse cross-blot hybridization have been recently reported for immediate identi¢cation of M. genavense infection in patients [20,21]. A thin layer chromatographic mycolic acid technique [1] and gas chromatographic fatty acid and mycolic acid cleavage product analysis [22] have also been used to identify M. genavense.…”

Section: Introductionmentioning

confidence: 99%

Isolation of a specific DNA fragment and development of a PCR-based method for the detection of Mycobacterium genavense

Chevrier

1999

FEMS Immunology and Medical Microbiology

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The rise of Mycobacterium genavense infections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734-bp fragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses speciesspecific primers for M. genavense. Followed by a non-radioactive hybridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7-kb sequence was specific for the M. genavense chromosome. The method developed here for M. genavense identification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated. z

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“…In oligonucleotide‐specific capture plate hybridization (OSCPH) the digoxigenin‐labeled amplicon is hybridized with a biotinylated M. genavense ‐specific oligonucleotide and captured on a streptavidin‐coated plate [19]. Amplification with biotinylated primers and analysis of the PCR products by reverse cross‐blot hybridization have been recently reported for immediate identification of M. genavense infection in patients [20, 21]. A thin layer chromatographic mycolic acid technique [1] and gas chromatographic fatty acid and mycolic acid cleavage product analysis [22] have also been used to identify M. genavense .…”

Section: Introductionmentioning

confidence: 99%

Isolation of a specific DNA fragment and development of a PCR-based method for the detection ofMycobacterium genavense

Chevrier

Oprişan

Maresca

et al. 1999

FEMS Immunology &Amp; Medical Microbiology

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The rise of Mycobacterium genavense infections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734-bp fragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses species-specific primers for M. genavense. Followed by a non-radioactive hybridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7-kb sequence was specific for the M. genavense chromosome. The method developed here for M. genavense identification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated.

show abstract

Routine Use of PCR–Reverse Cross-Blot Hybridization Assay for Rapid Identification of Mycobacterium Species Growing in Liquid Media

Cited by 31 publications

References 31 publications

Simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms by the reverse line blot hybridization assay

Simultaneous detection of multiple fly-borne bacterial pathogenic microorganisms by the reverse line blot hybridization assay

Isolation of a specific DNA fragment and development of a PCR-based method for the detection of Mycobacterium genavense

Isolation of a specific DNA fragment and development of a PCR-based method for the detection ofMycobacterium genavense

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