A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water.
As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either single-copy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation.
In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity.
Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 106 and 107 cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of target bacteria in samples are usually amplified by overnight pre-enrichment growth. These tests are usually performed in laboratories by skilled technicians. The present test was designed to overcome the shortcomings of the commercial hybridization tests.
The figure summarizes the major steps of the test. It is important to emphasize that the hybridization process used in this test differs from that of other hybridization tests in that it does not extract target nucleic acids. This process is based on intact-cell hybridization (so-called "in situ hybridization"), wherein the intact cells act as immobilizing agents. The cells are identified and enumerated by measuring the chemiluminescence emitted from alkaline phosphatase-probe (AP-probe) hybridization; the chemiluminescence is detected or measured by use of photographic film or a luminometer, respectively.
This test provides rapid, simple, and sensitive detection of microorganisms in water. The test is very flexible: specific probes can be developed for almost any group, genus, and, in many cases, species of microorganisms. The test can be performed in the field, or in a laboratory, using simple, battery-powered portable instrumentation. The test can be initiated and completed by non- technical persons. The test format can be automated easily. Probes for E.coli and the coliform group of bacteria have been developed for testing water. Results of a test can be obtained within 8 hours. Probes for Vibrio cholerae, Burk-holderia cepacia, Staphylococcus aureus, Staphylococcus epidermidis, and the Salmonella group have also been developed.
This work was done by Reinhardt A. Rosson, Julie Maurina-Brunker, Kim Langley, and Christine M. Pynnonen of Bio-Technical Resources, L.P. for Johnson Space Center. For further information, contact the Johnson Commercial Technology Office at (281) 483-3809. MSC-22663