ONPG test
The ONPG assay (abbreviation for. O - N itro p henyl- β - D - g alactopyranosid test) is a biochemical evidence for the enzyme β -galactosidase . The hydrolysis of the synthetic substrate o-nitrophenyl- β - D -galactopyranoside (ONPG) by a β- galactosidase is used in microbiological diagnostics to detect bacteria , for example from the genus Burkholderia , Salmonella and Nocardia , based on their β- galactosidase activity to distinguish from each other.
Basics
Some bacteria have the enzyme β-galactosidase, which enables them to metabolize the double sugar lactose . In the ONPG test, the synthetic galactoside o -nitrophenyl-β-D-galactopyranoside replaces the natural substrate of β-galactosidase. Like lactose, ONPG is brought into the cell by the galactosid permease and hydrolyzed by the β-galactosidase. This creates the two reaction products galactose and o -nitrophenol . The latter causes a yellow discoloration of the test solution in positive samples.
execution
For each sample, one ONPG disk and about 200 µl of sterile saline solution are added to a sterile tube so that the ONPG disk is completely under the sterile saline solution. Then about 1 to 3 colonies of the bacterium to be examined are suspended from a solid nutrient medium (agar plate / primary culture) with the aid of a sterile rod in the sterile saline solution. The tube with the mixture is then incubated at 36 ± 1 ° C. for a maximum of 24 hours and it is observed whether the suspension changes color.
evaluation
The reading of the test takes place after about 1, 6 and possibly 24 hours. A yellow coloration of the physiological saline solution indicates a positive ONPG test. Germs that do not show a positive color change after 6 hours should be incubated for up to 24 hours. If the yellow discoloration does not occur, this indicates a negative test result.
| ONPG positive bacteria | ONPG negative bacteria |
|---|---|
| Burkholderia cepacia | Burkholderia stabilis |
| Burkholderia multivorans | |
| Burkholderia vietnamiensis | |
| Burkholderia gladioli |
Since the ONPG test can lead to false positive results, an alternative detection of β-galactosidase with X-Gal is occasionally carried out when diagnosing staphylococci .
Individual evidence
- ^ R. De Ryck, MJ Struelens, E. Serruys: Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens. In: Journal of Clinical Microbiology . Volume 32, Number 6, June 1994, pp. 1583-1585, PMID 8077408 . PMC 264043 (free full text).
- ↑ M. Flores, EG Ford, JM Janda: Value of the O-nitrophenyl-beta-D-galactopyranoside test to differentiate among the aerobic actinomycetes. In: Journal of Clinical Microbiology. Volume 28, Number 9, September 1990, pp. 2142-2144. PMID 2121796 . PMC 268125 (free full text).
- ↑ H. Tse, E. Chan, CW Lam, KF Leung, P. Chow, KC Lee, KH Sze, SK Cheung, MK Tse, PL Ho, SP Leung, SK Lau, PC Woo, KY Yuen: Production of 2- aminophenoxazin-3-one by Staphylococcus aureus causes false-positive results in? -galactosidase assays. In: Journal of Clinical Microbiology. Volume 50, Number 11, November 2012, pp. 3780-3782, doi : 10.1128 / JCM.02299-12 . PMID 22972831 . PMC 3486264 (free full text).