DNA probes for infectious diseases

Clinical Microbiology Newsletter Vol. 7, No. 15 August 1, 1985

DNA Probes for Infectious Diseases

Fred C. Tenover, Ph.D. Associate Chief Microbiology Section Seattle Veterans Administration Medical

Center Seattle, Washington 98108

nology is actually simpler to perform than many routine immunofluorescent or ELISA methods. Herein, I will describe the preparation and use of DNA probes in a clinical microbiology laboratory.

A DNA probe is a tiny fragment of nucleic acid that can seek out and bind itself to other pieces of DNA which possess identical or nearly identical complementary sequences. DNA probes are very efficient at finding their complementary sequences, even in the presence of nonhomologous DNA from millions of other organisms. Because all microbial species have some unique DNA sequences that distinguish them from all other microorganisms, a DNA probe for virtually any pathogenic microorganism can be prepared using recombinant DNA techniques. The binding of. the probe to its complementary sequences can be observed and recorded by labeling the probe with a radiometric or colorimetric substrate. Labeled probes, when used to locate and identify specific microorganisms in clinical specimens, become highly specific and sen- sitive diagnostic tools, Because the probe binds directly to the DNA, the sensitivity of the test is unaffected by mutations which can often change the results of biochemical tests.

Probes are used in DNA-DNA hybridization reactions. Although per- forming D N A - D N A hybridization reactions to identify pathogens may seem to be beyond the capabilities of most clinical laboratories, the tech-

P r o b e D e v e l o p m e n t and Isolation Developing a DNA probe for a mi-

croorganism is usually a very long and tedious process. The highlights of the process are shown in Figure 1. The key is to identify the organism's "unique nucleotide sequence" and isolate it from the rest of the cell's DNA. This sequence may contain a specific .virulence gene, such as the enterotoxin gene of Escherichia coli, which Mose- ley and co-workers have used as a probe (12), or it may be a fragment of DNA cloned at random from the chro- mosome which may or may not have a known function. The genetic product or function of the probe is irrelevant to its ability to act as a useful diagnostic reagent. To isolate the unique sequence from the rest of the DNA, a set of enzymes, called restriction en- donucleases, are employed. The sequence (which is to become the DNA probe) is inserted into a plasmid or other type of cloning vector, and en- ters into a laboratory strain of E. coli by transformation. This organism will produce hundreds of copies of the recombinant plasmid containing the cloned sequence, making it possible to isolate large quantities of probe DNA. The plasmids are then isolated from E. coli, and the probe is separated from the plasmid and labeled so it can be

detected after hybridization. The last step is to thermally denature the DNA probe into single strands so that it can bind to its complementary sequences located in the genome of the organism to be identified.

P r inc ip l e of the D N A - D N A H y b r i d i z a t i o n Reaction

The basis of DNA probe technology is the D N A - D N A hybridization reaction. These reactions can be carried out with both the target and the probe DNA in solution, or, as is more common, with the DNA from the specimen immobilized on a solid phase, such as a nitrocellulose filter (Figure 2). This method, known as filter hybridization, was initially developed by Grunstein and Hogness (7), but later modified by Moseley and co- workers (12) to be more useful for

In T h i s Issue

DNA Probes . . . . . . . . . . . . . . . . . . . . . . 105 Technology for the rapid diagnosis of hlfectious diseases

Delta Virus . . . . . . . . . . . . . . . . . . . . . . . 108 Clinical, hnmunologic, and epidemiologic features of this newly recognized hepatotrophic virus

Parasitic Diseases . . . . . . . . . . . . . . . . 110 Intesthzal h~fection with multiple parasites

Abstract of Recent Li tera ture .. .111

NCCLS News . . . . . . . . . . . . . . . . . . . . . 111

CMNEEJ 7(15)105-112.1985 Elsevier 0196-4399/85/$0.00 + 02.20

I ~ Unique nucleotlde

sequence (probe]

Isolate DNA

Remove unique ~/JJ//~ sequence with

restriction enzyme

1

Insert sequence Into plasmld or cloning vector

#

[ ~ ~ 1 Transform plasmld into E Coil to make multiple copies of probe

#

~ Re Isolate probe sequences from

# ~ plasmlds

~ Label probe with Isotope or enzyme

Denature probe to single strands

Figure 1. Probe development and isolation.

clinical specimens. As shown in Figure 2, either organisms or clinical specimens can be inoculated onto the nitrocellulose filters. The samples are then treated with NaOH to lyse the organisms present and denature the DNA into single strands. Once the DNA is denatured, the filters are air dried and baked in a vacuum oven at 80°C in order to attach the DNA to the filter in a manner that leaves the single stranded bases available for hybridization with the labeled probe. When the complementary sequences of the probe and the target DNA come together, the labeled probe is bound and immobilized on the filter. After the filter is washed to remove nonspecifically bound probe DNA, the filter is tested for the presence of label. This technique has now been adapted for use with RNA viruses (9, 15); hepatitis B

Inoculate organisms or specimens to be tested on filter

I,

Treat filter to lyse organisms and denature DNA to single strands

Add label led probe to initiate hybridization reaction

Figure 2. Filter hybridization assay.

Wash filter to remove excess probe Add chromogenic substrate or perform autoradlography to Indicate probe binding (I.e., posit ive specimens)

virus (1, 10), genitourinary (4, 13, 17), stool (6, 12) and tissue specimens (18); and food samples (5, 8). Most investigators have used 32p-labeled nu- cleotides in a process known as nick translation (14)to label their probes. Although this method produces probes with very high specific activity, the short half-life of 32p (15 days) requires frequent labeling of the probe to achieve maximum sensitivity. Thus, the laboratory has to store large amounts of radioactive materials and generates a significant amount of radioactive waste.

To avoid the problems associated with the use of radioactive labels, an enzymatic method of labeling DNA was developed (1 1). This method ex- ploits the high affinity with which avidin, a protein found in egg whites, binds to biotin molecules. After incor- porating biotin into the nucelotide sequence of the probe by nick translation (a process that does not affect the probe's specificity), the probe can be

detected by exposing the hybridized DNA to avidin which has been conju- gated to one of geveral enzymes such as alkaline phosphatase, acid phosphatase, or horseradish peroxidase. The addition of a chromogenic substrate to the reaction mix allows the double- stranded DNA present to be detected colorimetrically. Enzymatically labeled probes that have a shelf life of 1 yr have been used successfully by both Sixbey et al. (I6) and Brigatti et al. (3). At present, the radiometrically labeled probes appear slightly more sen- sitive than their enzymatically labeled counterparts; however, well-controlled comparative studies using clinical specimens have not been reported.

Ex a mp l e s o f DNA Probes The list of DNA probes that have

been used for detecting infectious agents is shown in Table 1. A review of each of these probes is beyond the scope of this article; however, I would like to comment specifically on the use

106 0196-4399/85/$0.00 + 02.20 © 1985 Elsevier Science Publishing Co., Inc. Clinical Microbiology Nev, sletter 7:15,1985

of DNA probes for identifying viruses and stool pathogens.

DNA probes could have a tremen- dous impact on identifying viral agents in clinical specimens. Table 1 lists several of the DNA probes that have been described for viral agents. Many of these agents cannot be cultivated routinely in vitro (e.g., hepatitis B virus), or can take as long as 6 weeks to manifest cytopathic effect in cell lines, (e.g., cytomegalovirus). The need for setting up a variety of expensive cell cultures can be reduced by identifying the presence of viruses directly in patient specimens. Thus, DNA probes should be able to de- crease the time necessary for the detection of the infectious agent as well as reduce the cost of specimen processing. In this regard, Chou and Merrigan (4) showed that DNA probes could be used to detect the often slow- growing cytomegalovirus (CMV) directly in urine. Although their proce- dure required ultracentrifugation of the specimen, making it impractical for most clinical laboratories, the study demonstrates that a DNA probe can be used for rapid detection of CMV in clinical specimens. Because the hybridization technique itself is simple to perform, with some modification, this technique could be instituted in many laboratories.

The studies of Redfield et al. (13), which describe the development of probes for herpes simplex viruses I and II, suggest that DNA probes for these viruses may soon be a diagnostic reality. These investigators detected as few as four HSV-I and eight HSV- II infected cells in clinicaI specimens. Although they used a radiolabeled probe that required 24 hr of processing, these investigators state that a biotinylated probe would permit this test to be completed in approximately 6 hr from the time the specimen is re- ceived in the laboratory.

The studies of Flores et al. (6) with DNA probes for rotaviruses, Hyypia et al. (9) and Rotbart et al. (15) with probes for enteroviruses, and Ber- ninger et al. (1) and Kam et al. (10) who developed DNA probes for hepatitis B virus, all support the utility of probes in the clinical laboratory.

Table 1 DNA Probes for Infectious Agents

1. Enterotoxigenie E. coli 2. Yersinia enterocolitica 3. Sabnonella spp. 4. Shigella spp. 5. Neisseria gonorrhoeae 6. Mobihmcus spp. 7. Leishmania mexicana 8. Leishmania braziliensis 9. Herpes simplex virus I and II

10. Cytomegalovirus 11. Entcroviruses 12. Epstein-Barr virus 13. Hepatitis B virus 14. Papilloma virus 15. Adenovirus 16. Varicella zoster virus 17. Human T-cell leukemia virus 18. Rotavirus

The isolation and identification of pathogenic bacteria from stool is just as expensive and time consuming as viral isolation. Many laboratories cur- rently use four or more selective media to screen for such organisms in a process that can take as long as 96 hr. However, if specimens were initially screened with DNA probes, and only those demonstrating a positive result cultured, a diagnosis could often be made in less than 6 hr and the cost of processing the stool culture might be reduced by as much as 50%. DNA probes have been described for salmo- nella (5), shigella (2), and Yersinia en-

terocolitica (8). Therefore, such sav- ings might be realized in the near fu- ture, provided that a rapid method of probing stools can be developed. Even with a rapid detection system, in some cases, the specimen will need to be cultured and the organism isolated in order to perform serotyping and an- timicrobial susceptibility testing.

A d v a n t a g e s of D N A P r o b e s DNA probes offer the advantages of

rapid organism identification with the use of the biotin/avidin system, the po- tential to reduce costs by avoiding expensive culture techniques, and the feasibility of using batteries of probes to screen specimens before culture. A battery of probes could be particularly valuable for screening specimens such as cerebral spinal fluid, sputum, and stool where the presence or absence of a select group of organisms could rapidly be determined. Probes also react well with nonviable organisms, an important consideration for many viral agents, which are often thought to be lost during transport. Finally, the biotinylated probes have a shelf life of 1

yr, much longer than many biochemical media.

D i s a d v a n t a g e s of D N A P r o b e s The disadvantages of DNA probes

in clinical microbiology laboratories include the current emphasis on ra- dionucleotides for signal detection, the low sensitivity of probes in highly pro- teinaceous specimens, and the general lack of expertise for interpreting re- suits. The majority of microbiologists will not want to adopt a radiometric identification method, primarily because of the regulatory problems associated with maintaining radioactive elements in a laboratory. Thus, DNA probes will probably not be widely used in clinical laboratories until the nonradiometric detection methods become available.

The most important question that has yet to be answered concerns the sensitivity of DNA probes when used on clinical specimens. Although many of the probes described in the literature appear to exhibit high sensitivity, often they have only been tested against pure cultures of organisms. A variety -of well-controlled clinical trials for this new technology is needed.

The lack of technologist expertise in this field is a disadvantage that will be resolved only with training and experi- ence, much as with the introduction of fluorescent-labeled antibody tests several years ago. DNA hybridization reactions are, however, simpler to perform than fluorescent tests, and in the long run will probably be less expensive on a per test basis.

Finally, DNA probe kits for diagnostic purposes are not yet commer- cially available. DNA probe kits are available, however, on a research basis

Clinical Microbiology Newsletter 7:15,1985 © 1985 Elsevier Science Publishing Co., Inc. 0196-4399"85/$0.00 + 02.20 107

from Enzo Biochemicals, Inc., New York, NY, and will be available shortly from Gen-Probe, San Diego, CA.

Conclus ions DNA probes will have a significant

impact on the field of clinical microbiology and will broaden the range of organisms that can be identified. This technology will provide a highly specific means of rapidly diagnosing a variety of bacterial, viral, and parasitic infections that previously have gone undetected.

References 1. Berninger, M. M. et al. 1982. An

assay for the detection of the DNA genome of hepatitis B virus in serum. J. Med. Virol. 9:57-68.

2. Boileau, C. R., H. M. d'Hauteville, and P. J. Sansonetti. 1984. DNA hybridization technique to detect Shigella species and enteroinvasive Escherichia coli. J. Clin. Microbiol. 20:959-961.

3. Brigatti, D. J. et al. 1982. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labelled hybridization probes. Virology 126:32-50.

4. Chou, S., and T. C. Merrigan.

1983. Rapid detection and quantitation of human cytomegalovirus in urine through DNA hybridization. N. Engl. J. Meal. 308:921-925.

5. Flits, R. et al. 1983. DNA-DNA hybridization assay for detection of Sal- monella spp. in foods. Appl. Environ. Microbiol. 46:1146-1151.

6. Flores, J. et al. 1983. A dot hybridization assay for detection of rotavirus. Lancet i:555-559.

7. Grunstein, M., and D. S. Hogness. 1975. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72:3961-3965.

8. Hill, W. E., W. L. Payne, and C. C. G. Aulisio. 1983. Detection and enumeration of virulent Yersbffa enterocolitica in food by DNA colony hybridization. Appl. Environ. Microbiol. 46:636-641.

9. tlyypia, T. el al. 1984. Detection of enteroviruses by spot hybridization. J. Clin. Microbiol. 19:436-438.

10. Kam, W. et al. 1982. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc. Natl. Acad. Sci. USA 79:7522-7526.

11. Leary, J. J. , D. J. Brigati, and D. C. Ward. 1983. Rapid and sensi- tive colorimetric method for visual- izing biotin labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bio-blots. Proc. Natl. Acad. Sci. USA 80:4045-4049.

12. Moseley, S. L. et al. 1980. Detection of cntcrotoxigenic Escherichia coli by DNA colony hybridization. J. Infect. Dis. 142:892-898.

13. Redfield, D. C. et al. 1983. Detection of herpes simplex virus in clinical specimens by DNA hybridization. Diagn. Microbiol. Infect. Dis. 1:I 17- 128.

14. Rigby, P. W. J. et al. 1977. La- belling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251.

15. Rotbart, H. A., iM. J. Levin, and L. P. Villarreal. I984. Use of subgcnomic poliovirus DNA hybridization probes to detect major subgroups of enteroviruses. J. Clin. Microbiol. 20:1105-1108.

16. Sixbey, J. W. et al. 1984. Epstein- Barr virus replication in oropharyngeal epithelial cells. N. Engl. J. Med. 310:1225-1230.

17. Totten, P. A. et al. 1983. DNA hybridization technique for the detection of Neisseria gonorrhoeae in men with urethritis. J. Infect. Dis. 148:462- 471.

18. Wirth, D. F., and D. M. Pratt. 1982. Rapid identification of Leish- mania species by specific hybridization of kinetoplast DNA in cutaneous le- sions. Proc. Natl. Acad. Sci. USA 79:6999-7003.

Editorial

Delta Hepatitis

Stephen C. Hadler, M.D. Chief, Epidemiology Activity Hepatitis Branch Division of Viral Diseases Center for Infectious Diseases Centers for Disease Control Atlanta, Georgia 30333

Originally described in 1977 by Riz- zetto as a new hepatitis B virus (HBV) antigen/antibody system, the delta agent has now been characterized as a distinct hepatotrophic virus. Its impor- tance lies in two features. First, it is a unique human pathogen that is able to cause infection only in the presence of another hepatitis virus (hepatitis B), and second, it is able to modify the outcome of hepatitis B infection, aug-

menting the severity of acute and chronic HBV disease. Thus, delta agent is a current focus of intensive research throughout the world.

B i o l o g y The delta virus is a 3 5 - 3 7 nm par-

ticle with three components: a 500,000 mw single strand of RNA; a 62,000 mw internal protein antigen, the delta antigen; and, an external protein " c o a t , " the hepatitis B surface antigen. Although the virus borrows its " c o a t " from the hepatitis B virus, its RNA shows no homology with hepatitis B DNA, and the delta antigen shares no epitopes with those of hepatitis B antigens.

H u m a n Diseases The delta virus depends on HBV for

replication and can produce infection

only in the presence of active HBV infection. Infection with delta agent may occur in two situations----coinfection with hepatitis B virus (HBV) and superinfection of an HBV carrier. Coinfection occurs when a person sus- ceptible to HBV is exposed to blood that contains both the hepatitis B and delta viruses, whereas superinfection results when an HBV carrier is exposed to blood containing the delta virus. Each infection results in an epi- sode of acute hepatitis that is clinically indistinguishable from that due to other hepatitis viruses. In coinfection, acute hepatitis develops after an incu- bation period of 4 - 2 0 weeks, and is characterized by initial replication of HBV and subsequent replication of delta virus. The usual course of coinfection is resolution of both HBV and delta infection; probably fewer than

108 0196-4399/85/$0.00 + 02.20 © 1985 Elsevier Science Publishing Co., Inc. Clinical Microbiology Newsletter 7:15,1985

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DNA probes for infectious diseases