DETECTION AND QUANTITATION OF RED COMPLEX BACTERIA IN SUBGINGIVAL PLAQUE BY USING FLUORESCENT IN SITU HYBRIDIZATION (FISH)

Motivation/Background: Red complex bacteria are proven periodontal pathogens. In dentistry, there is a need to identify and quantitate the organisms from the diseased sites quickly and reliably. Since culture requires several days, molecular methods are being used frequently to detect these bacteria. Among them, Fluorescent in situ hybridization (FISH) is rapid, sensitive and quantitative. An attempt is made here to evaluate the applicability of this technique as a diagnostic tool in periodontology. Method: Subgingival plaque was collected from participants, fixed with paraformaldehyde and subjected to FISH. Fluorescently labeled oligonucleotide probes were used for hybridization. After the procedure, the fluorescently stained bacteria were identified and counted from the smear and quantitated using a simple grading. Results: There was a significant difference in the prevalence and numbers of red complex bacteria in healthy and diseased subjects. A strong linear relationship existed between P. gingivalis, T. forsythia and T. denticola. Conclusions: The procedure used in the study is simple, rapid and can be easily adaptable. It also has a high sensitivity and has the ability to detect a single bacterial cell. The method can be directly applied to the clinical samples and can be used as a rapid diagnostic tool in periodontics.

Download Full-text

Fluorescent in situ hybridization with specific DNA probes offers adequate detection of Enterococcus faecalis and Enterococcus faecium in clinical samples

Journal of Medical Microbiology ◽

10.1099/jmm.0.46022-0 ◽

2005 ◽

Vol 54 (10) ◽

pp. 937-944 ◽

Cited By ~ 39

Author(s):

Karola Waar ◽

John E Degener ◽

Marja J van Luyn ◽

Hermie JM Harmsen

Keyword(s):

In Situ Hybridization ◽

Enterococcus Faecalis ◽

Enterococcus Faecium ◽

Fluorescent In Situ Hybridization ◽

Dna Probes ◽

Clinical Samples

Download Full-text

Fluorescent in situ hybridization (FISH) as a diagnostic tool for Williams-Beuren syndrome

Genetics and Molecular Biology ◽

10.1590/s1415-47572007000100005 ◽

2007 ◽

Vol 30 (1) ◽

pp. 17-20 ◽

Cited By ~ 6

Author(s):

Deise Helena de Souza ◽

Danilo Moretti-Ferreira ◽

Lígia Maria Suppo de Souza Rugolo

Keyword(s):

In Situ Hybridization ◽

Diagnostic Tool ◽

Fluorescent In Situ Hybridization

Download Full-text

Diagnostic value comparison of CellDetect, fluorescent in situ hybridization (FISH), and cytology in urothelial carcinoma

Cancer Cell International ◽

10.1186/s12935-021-02169-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Donghao Shang ◽

Yuting Liu ◽

Xiuhong Xu ◽

Zhenghao Chen ◽

Daye Wang

Keyword(s):

Bladder Cancer ◽

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Urine Cytology ◽

Low Grade ◽

High Grade ◽

Muscle Invasive Bladder Cancer ◽

Significant Difference ◽

Muscle Invasive

Abstract Background To evaluate the clinical effectiveness of a novel CellDetect staining technique, compared with fluorescent in situ hybridization (FISH), and urine cytology, in the diagnosis of urothelial carcinoma (UC). Methods A total of 264 patients with suspicious UC were enrolled in this study. All tissue specimens were collected by biopsy or surgery. Urine specimen was obtained for examinations prior to the surgical procedure. CellDetect staining was carried out with CellDetect kit, and FISH was performed with UroVysion detection kit, according to the manufacturer’s instructions. For urine cytology, all specimens were centrifuged using the cytospin method, and the slides were stained by standard Papanicolaou stain. Results In this study, there were 128 cases of UC and 136 cases of non-UC, with no significant difference in gender and age between the two groups. Results for sensitivity of CellDetect, FISH, and urine cytology were 82.8%, 83.6%, and 39.8%, respectively. The specificity of the three techniques were 88.2%, 90.4%, and 86.0%, respectively. The sensitivity of CellDetect and FISH are significantly superior compared to the conventional urine cytology; however, there was no significant difference in specificity among three staining techniques. In addition, the sensitivity of CellDetect in lower urinary tract UC, upper urinary tract UC, non-muscle-invasive bladder cancer (NMIBC), and muscle-invasive bladder cancer (MIBC) were 83.3%, 81.8%, 83.5%, and 72.0%, respectively. The screening ability of CellDetect has no correlation with tumor location and the tumor stage. The sensitivity of CellDetect in low-grade UC and high-grade UC were 51.6 and 92.8%. Thus, screening ability of CellDetect in high-grade UC is significantly superior compared to that in low-grade UC. Conclusions CellDetect and FISH show equal value in diagnosing UC, both are superior to conventional urine cytology. Compared to FISH, CellDetect is cost effective, easy to operate, with extensive clinical application value to monitor recurrence of UC, and to screen indetectable UC.

Download Full-text

Specific and Rapid Detection by Fluorescent In Situ Hybridization of Bacteria in Clinical Samples Obtained from Cystic Fibrosis Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.38.2.818-825.2000 ◽

2000 ◽

Vol 38 (2) ◽

pp. 818-825 ◽

Cited By ~ 105

Author(s):

Michael Hogardt ◽

Karlheinz Trebesius ◽

Anna M. Geiger ◽

Mathias Hornef ◽

Josef Rosenecker ◽

...

Keyword(s):

Cystic Fibrosis ◽

In Situ Hybridization ◽

Rapid Detection ◽

Burkholderia Cepacia ◽

Fluorescent In Situ Hybridization ◽

Clinical Samples ◽

Microbial Culture ◽

Specific Detection ◽

Pulmonary Exacerbations

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia,Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 105 CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, andP. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

Download Full-text

Analysis of numerical aberrations in specific chromosomes by fluorescent in situ hybridization as a diagnostic tool in breast cancer

Cancer ◽

10.1002/(sici)1097-0142(19960515)77:10<2064::aid-cncr15>3.0.co;2-t ◽

1996 ◽

Vol 77 (10) ◽

pp. 2064-2069 ◽

Cited By ~ 56

Author(s):

Daisuke Ichikawa ◽

Naoya Hashimoto ◽

Masakazu Hoshima ◽

Toshiharu Yamaguchi ◽

Kiyoshi Sawai ◽

...

Keyword(s):

Breast Cancer ◽

In Situ Hybridization ◽

Diagnostic Tool ◽

Fluorescent In Situ Hybridization ◽

Numerical Aberrations

Download Full-text

Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

Applied and Environmental Microbiology ◽

10.1128/aem.68.9.4225-4232.2002 ◽

2002 ◽

Vol 68 (9) ◽

pp. 4225-4232 ◽

Cited By ~ 90

Author(s):

Erwin G. Zoetendal ◽

Kaouther Ben-Amor ◽

Hermie J. M. Harmsen ◽

Frits Schut ◽

Antoon D. L. Akkermans ◽

...

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

16S Rrna ◽

Fluorescent In Situ Hybridization ◽

Specific Effect ◽

Human Intestine ◽

Fecal Samples ◽

Flow Cytometric ◽

Significant Difference

ABSTRACT A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.

Download Full-text