DNase Test

Principle

The deoxyribonuclease (DNase) test detects the degradation of DNA by bacterial species that produce DNase. The DNase test may be performed on plate media and is available in some commercial tests, e.g., QuadFERM+. When DNase is produced by organisms in the QuadFERM+ test, an acidic endproduct is formed and the pH indicator changes from red (alkaline) to yellow (acid) e.g., Figure 1.

Figure 1. Example of a Rapid DNase Test for Neisseria and Related Species.

Specimen Requirements

Optimum specimen: Pure culture (18-24 h.) of gram-negative, oxidase-positive, catalase-positive diplococci grown on nonselective (chocolate or equivalent) medium isolated either from selective (Modified Thayer-Martin; MTM) medium or nonselective (chocolate or equivalent) medium.

Unacceptable specimens:

• Contaminated cultures grown on nonselective medium
  
• Apparently pure cultures grown on selective medium
  

• Pure cultures older than 18-24 h. grown on nonselective medium

Factors that compromise test results:

• Use of contaminated cultures to inoculate test.
  
• Incubation of tests in an incubator with carbon dioxide-supplemented atmosphere. Carbon dioxide may diffuse into the medium and form carbonic acid which may cause a false-positive acid reaction.

Stability of specimen:

• The DNase test is dependent on preformed enzymes. Test should be inoculated within 30 min. of removing culture from incubator; prolonged exposure at room temperature may result in diminished enzyme activity.

Medium/Reagents

DNase tests may be performed on plate media which incorporate indicators. However, most media for DNase tests do not support the growth of fastidious Neisseria species.

QuadFERM+: Commercial, an acid detection test, includes a DNase test.

Store in accordance with manufacturer's directions.

Quality Control/Test Procedure

Quality control of the DNase test is performed concurrently with QC of the acid detection component of the QuadFERM+ test.

QC strains:

• Negative control: N. gonorrhoeae, strain ATCC 31426

• Positive control: M. catarrhalis, strain ATCC 25240

  QC strains are stored at -70C in a solution of trypic soy broth containing 20% glycerol. Reactions of QC strains should be confirmed at the time the frozen stocks are prepared. QC strains may be stored at -70C for up to 2 years.

Procedure:

1. Thaw vials of QC strains stored at -70C. Clinical isolates may be subcultured from selective medium or purified subcultures.
2. Streak-inoculate strains onto plates of chocolate agar (or equivalent medium) for isolation. Incubate plates at 35C to 36.5C in a carbon dioxide-enriched (5%) atmosphere for 18 to 24 h. Confirm that cultures are pure.

3. Perform tests as described in the manufacturer's directions. The results for the Quality Control strains are shown in Table 1.

   Positive reaction is recorded when the color in the test medium is more orange/yellow than the color in the control medium.
   Negative reaction is recorded when the color in the test medium is the same, or a darker red, than the color in the control medium.

4. Read and record results.

Table 1. DNase Production Patterns of Quality Control Strains of N. gonorrhoeae and M. catarrhalis.

QC Strain	DNase Reaction
N. gonorrhoeae, ATCC 31426 DNase-negative
M. catarrhalis, ATCC 25240 DNase-positive

Quality Control Schedule:

• Quality control tests should be performed on each new lot of quadFERM+ upon receipt from the manufacturer.
  
• Note: If more than one lot number is received at the same time quality control testing must be performed on each lot.
  

• Note: If two shipments of the same lot numbers are received at different times, quality control tests must be performed on test strips from each shipment.

Table 2. DNase Reactions of Neisseria and Related Species.

The control cupule containing no carbohydrate remains red (no acid has been produced). Cupules with contents that are more yellow than the contents in the control cupule are recorded as positive.

DNase Reaction	Species
Positive M. catarrhalis N. ovis (sheep isolate)
Negative N. gonorrhoeae N. meningitidis N. lactamica N. kochii N. cinerea N. polysaccharea N. flavescens N. mucosa N. subflava biovars subflava/flava N. subflava biovar perflava N. sicca N. elongata K. denitrificans

Problems & Solutions

Incubation of the strips in a incubator with an enhanced carbon dioxide atmosphere may cause false-positive reactions because carbon dioxide will dissolve in the media and form carbonic acid, a weak dibasic acid which, in turn, will cause an acid reaction in the medium. Incubate tests only in incubators without supplemental carbon dioxide.

Strips must be warmed to room temperature before use. The test relies on preformed enzymes. Inoculation of cold test strips may reduce the activity of the enzymes

Saline containing preservatives, buffers, or bacteriostatic agents should NOT be used in preparing bacterial suspensions; these may denature the preformed enzymes that react with the substrates.

Do not make bacterial suspensions in water; enzymes may be denatured.

Use pure cultures only. Mixed cultures may include organisms that produce acid from carbohydrates different from those of the test neisserial strain.

Use bacterial suspensions immediately (within 15 min after preparation from cultures that have been held at room temperature no longer than 30 min). Enzymes may denature if suspensions are not used immediately.

If many strains are to be tested, inoculate each test strip immediately after preparation of the suspension rather than preparing all suspensions and then inoculating tests; this inoculation strategy will also minimize the possibility of mislabeling a test strip.

Limitations of Test

The DNase is used only as a supplemental test to aid in the identification of Neisseria and related species which have been characterized in other tests such as acid detection tests.

The DNase test distinguishes M. catarrhalis from all other gram-negative diplococci of human origin. Only N. ovis, a species isolated from sheep and cattle, produces DNase and fails to produce detectable acid from carbohydrates. It should be noted that strains of N. ovis have occasionally been isolated from humans. The colonies of N. ovis are translucent and butyrous compared with those of M. catarrhalis which are opaque and usually friable.

The DNase test may not be used to differentiate between other Neisseria and related species.

Results, Interpretation, & Reporting

The DNase is used only as a supplemental test to aid in the identification of Neisseria and related species which have been characterized in other tests such as acid detection tests.

Bibliography

Bovre K. 1984. Family VIII. Neisseriaceae Prevot, p. 288-309. In Krieg NR (ed.). Manual of Systematic Bacteriology, vol. 1. The Williams & Wilkins co., Baltimore.

Knapp JS. Historical perspectives and identification of Neisseria and related species. Clin Microbiol Rev 1988;1:415-431.

Vedros NA. 1984. Genus I. Neisseria Trevisan 1885, 105AL, p. 290-296. In Krieg NR (ed.). Bergey's Manual of Systematic Bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore.