Genetic fingerprinting of Theileria parva using a telomeric DNA probe

 A cloned Theileria parva telomeric DNA sequence, designated pTpUtel, was used to characterize T. parva stocks and clones by hybridization to EcoRI-digested DNA. Eight of the nine T. parva stocks tested were discriminated by the telomeric restriction-fragment-length polymorphisms (RFLPs). Two isolates derived from buffalo 7014 by tick feeding on different occasions were also differentiated using the probe. The probe gave comparable results on purified piroplasm or schizont-infected lymphocyte DNA and did not cross-hybridize with uninfected bovine DNA. The telomeric restriction pattern of a cloned T. parva parasite remained identical after four passages through ticks and cattle. The telomeric sequence therefore represents a useful additional tool for analysis of theileriosis epidemiology. Received: 25 May 1995 / Accepted: 25 August 1995     

Cloning and characterization of a complex DNA fingerprinting probe for Candida parapsilosis

Candida parapsilosis accounts for a significant number of nosocomial fungemias, but in fact, no effective and verified genetic fingerprinting method has emerged for assessing the relatedness of independent isolates for epidemiological studies. A complex 15-kb DNA fingerprinting probe, Cp3-13, was therefore isolated from a library of C. parapsilosis genomic DNA fragments. The efficacy of Cp3-13 for DNA fingerprinting was verified by a comparison of its clustering capacity with those of randomly amplified polymorphic DNA analysis and internally transcribed spacer region sequencing, by testing species specificity, and by assessing its capacity to identify microevolutionary changes both in vitro and in vivo. Southern blot hybridization of EcoRI/SalI-digested DNA with Cp3-13 provides a fingerprinting system that (i) identifies the same strain in independent isolates, (ii) discriminates between unrelated isolates, (iii) separates independent isolates into valid groups in a dendrogram, (iv) identifies microevolution in infecting populations, and (v) is amenable to automatic computer-assisted DNA fingerprint analysis. This probe is now available for epidemiological studies.     

PCR-based DNA fingerprinting of Staphylococcus haemolyticus to investigate nosocomial infections

Objective: To apply PCR-based DNA fingerprinting in a clinical microbiology laboratory to investigate nosocomial infections with Staphylococcus haemolyticus.
Method: DNA fingerprints were generated by PCR on 99 S. haemolyticus isolates using different primer combinations based on ERIC, REP or arbitrarily chosen simple repeat sequences.
Results: Primer combinations REP1+(GTC)₆ and ERIC1+ERIC2 had sufficient discrimatory power and were chosen to analyze the clinical isolates. DNA fingerprint patterns from strains isolated from the patients nursed in the same hospital ward in the period 1991–94 were approximately 90% similar to each other. One staff member, sampled in 1991, carried a strain with a similar fingerprint.
Conclusions: PCR based DNA fingerprinting is a suitable method to perform in a clinical laboratory. An S. haemolyticus strain appeared to be endemic in the hospital ward and had most probably been transmitted from patient to patient. S. haemolyticus may carry glycopeptide resistance and needs attention as a causative agent of nosocomial infections.     

Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.     

Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting.

The gastric pathogen Helicobacter pylori establishes long-term chronic infections that can lead to gastritis, peptic ulcers, and cancer. The species is so diverse that distinctly different strains are generally recovered from each patient. To better understand the dynamics of long-term carriage, we characterized H. pylori isolates from initial and follow-up biopsy specimens from a patient population at high risk of H. pylori infection and gastric cancer. Eighty-five isolates were obtained from 23 patients and were analyzed by genomic restriction enzyme analysis, arbitrarily primed PCR fingerprinting, (random amplified polymorphic DNA analysis), and/or restriction of specific PCR-amplified genes (restriction fragment length polymorphism analysis). A single strain was found in sequential biopsy specimens from 12 of 15 patients (80%) receiving sucralfate. In the remaining three patients treated with sucralfate, two strains were identified in two patients and three strains were identified in the third patient. In contrast, a single strain was found in sequential biopsy specimens from only three of eight patients (37%) receiving bismuth, metronidazole, and nitrofurantoin. Two strains were identified in five other patients receiving bismuth-antibiotic (63%). Immunoglobulin G antibodies to H. pylori were present in the sera of all patients. Thus, H. pylori colonization can persist for long periods (up to at least 4 years), despite high titers of immunoglobulin G antibodies in serum. Resistance to metronidazole was noted in some strains before and/or after treatment, but all strains remained susceptible to amoxicillin, tetracycline, and nitrofurantoin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of unrelated strains of Staphylococcus schleiferi by using ribosomal DNA fingerprinting, DNA restriction patterns, and plasmid profiles.

The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species.     

DNA fingerprinting in a random sample of Japanese population with the minisatellite probe MZ 1.3

The DNA fingerprinting analysis with minisatellite probe MZ 1.3 with a nonisotopic technique is described. We determined a random sample of Japanese population with 36 samples.     

DNA fingerprinting by pulsed field gel electrophoresis and ribotyping to distinguish Pseudomonas cepacia isolates from a nosocomial outbreak.

We typed 40 isolates of Pseudomonas cepacia obtained from patients involved in a single outbreak using pulsed field gel electrophoresis and ribotyping. All isolates from the majority of the patients, 16 of 18 (89%), were included in a single group. These typing methods should aid in the clarification of the epidemiology of infection with P. cepacia.     

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence.

We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10(8) copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test.     

Fluorescence-based DNA fingerprinting elucidates nosocomial transmission of phenotypically variablePseudomonas aeruginosa in intensive care units

DNA fingerprinting based on automated laser fluorescence analysis of randomly amplified polymorphic DNA (RAPD-ALFA) is a rapid and convenient technique for detecting clonal relatedness of bacterial isolates of nosocomial concern. During an outbreak ofPseudomonas aeruginosa among five patients in a medical intensive care unit, transmission was not suspected because of the phenotypic variability of the initial isolates. However, DNA fingerprinting by RAPD-ALFA and macrorestriction analysis identified a single genotype (strain A) for isolates from three patients and another genotype (strain B) for isolates from the remaining two patients. Strain A isolates displayed three phenotypes defined by different antibiotypes and distinct colony appearance. Retrospective analysis of DNA fingerprints demonstrated that strain A had been transmitted to the index patient one year previously in a different intensive care unit. The study demonstrates that genetic typing approaches are warranted should epidemiological relatedness be identified between phenotypically variant pathogens. Automated laser fluorescence analysis of PCR fingerprints may facilitate routine screening of bacterial isolates for in-house epidemiological surveillance. Antibiograms are an unsuitable approach for the typing ofPseudomonas aeruginosa.     

In situ hybridisation in herpetic lesions using a biotinylated DNA probe.

In situ hybridisation was performed with a biotinylated DNA probe for herpes simplex virus (HSV) using high temperature denaturation on formalin fixed, paraffin wax sections of lung, brain, ganglion and keratinising and non-keratinising squamous epithelia. Eosinophilic viral nuclear inclusions or characteristically moulded multiple nuclei with altered chromatin, which were present in two cases of HSV encephalitis and one case of viral pneumonitis, all showed complete hybridisation visualised by an alkaline phosphatase/nitroblue tetrazolium detector system. HSV encephalitis and trigeminal ganglionitis, which were confirmed serologically or clinicopathologically but lacked nuclear changes, also gave positive dense nuclear signal in neurons, glias and satellite cells. No staining was present in the ganglion cells in trigeminal zoster, the glia in progressive multifocal leucoencephalopathy, or in a variety of cells in a lung coinfected with cytomegalovirus. In 10 herpetic blisters of squamous epithelia, infected cells hybridised strongly, while morphologically similar herpes zoster lesions remained negative. In neural tissues non-hybridisation staining was most obtrusive in corpora amylacea and seemed to reflect nonspecific probe adherence. In squamous epithelium, major non-hybridisation staining was caused by probe and antibody possibly adhering to intracellular keratin. The HSV probe permits specific detection of virus in the absence of characteristic nuclear changes and allows varicella zoster virus to be differentiated from HSV, provided that the aforementioned problems with non-hybridisation staining are borne in mind.     

Development of a diagnostic test for Johne''s disease using a DNA hybridization probe.

A DNA probe, M13 mpHAW71, that detects Mycobacterium paratuberculosis in the fecal material of infected animals was developed for use in the diagnosis of Johne's disease. The probe detected as few as 10(5) M. paratuberculosis when hybridized under stringent conditions to total genomic DNA purified from bovine fecal material. When the probe was used diagnostically, it did not differentiate members of the Mycobacterium avium-M. intracellulare-M. paratuberculosis complex. Compared with culturing, the DNA probe identified 34.4% more mycobacterium-containing fecal samples, and testing took only 72 h to complete.     

DNA fingerprinting of Mycobacterium tuberculosis isolates from Agra region by IS 6110 probe

DNA fingerprinting using IS 6110 probe has been used all over the world quite successfully to characterize M. tuberculosis strains. The present study has been carried out to study the polymorphism among isolates of M. tuberculosis from Agra region from patients attending the clinics at SN Medical College and TBDTC, Agra. Sputa were collected in sterilized containers and brought to CJIL, Agra. Samples were processed and cultured on Lowenstein Jensen (LJ) slants. M. tuberculosis isolates were identified by standard biochemical tests. DNA from these isolates were purified by a physicochemical procedure, restricted with Pvu II enzyme and hybridized with PCR amplified and DIG labeled 245 bp IS 6110 probe. With a view to study IS 6110 polymorphism, M. tuberculosis isolates obtained from different geographical areas of Agra region were analyzed. Among the 60 isolates taken in study, 5 had no copy of IS 6110, 8 had 1-4 copies and 47 had multiple copies of IS 6110. DNA fingerprinting using this probe was found to be quite discriminating for typing of most of the strains (80%) which had multiple copies. RFLP profiles did not correlate with geographical areas, contacts or the resistance pattern of the strains. While this data shows the potential of IS 6110 based RFLP for strain characterization of M. tuberculosis in Agra, to understand the molecular epidemiology of tuberculosis in this region, a larger number of isolates from defined geographical areas need to be studied.     

Characterization and sequence of lupin mitochondrial plasmid-like DNA

Summary Two minicircular DNAs of 1.2 kb (K1) and 1.4 kb (K2) were found in mitochondria of fertile lupin (Lupinus albus). The plasmid-like DNA, K1, was cloned, labelled and hybridized with mitochondrial DNA from three different species of lupin. We have found no evidence for integrated copies of K1 in any of the mitochondrial genomes probed in this study. No sequence homology between plasmid K1 and K2, and no homology of either with chloroplast DNA, has been detected. The K1 DNA is two-fold more abundant than the K2 DNA and about seven-fold more abundant than a unique segment of the mtDNA. The entire nucleotide sequence of the K1 DNA has been determined. This sequence exibits a 340 base pair region with highly organized repeats. The sequence of K1 shows no substantial homology with sequence of other mitochondrial plasmids of higher plants.     

Development of a DNA probe for Streptococcus bovis by using a cloned amylase gene.

Streptococcus bovis is a normal inhabitant of the rumen but has been implicated as a causative agent for ruminal lactic acidosis and related problems. While rarely isolated from humans, S. bovis has been identified as a causative agent for endocarditis, meningitis, and septicemia. Recent reports have also suggested a correlation between human colonic carcinoma and increased levels of S. bovis. Identification of S. bovis strains of human origin has been problematic because of variations in results of biochemical tests compared with results for ruminal strains. We have tested a cloned amylase gene from the ruminal strain S. bovis JB1 as a potential DNA probe for rapid and accurate identification of S. bovis strains from all sources. DNAs from strains identified as S. bovis, of both human and ruminal origin, were found to hybridize with the probe under stringent conditions. The probe also hybridized with variants of S. bovis that did not grow on starch. The probe did not hybridize with DNA isolated from other bacteria of human colonic and ruminal origin, including Bacteroides thetaiotaomicron, Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Enterococcus faecalis but did demonstrate hybridization with Streptococcus salivarius.     

Evaluation of a Salmonella-specific DNA probe by colony hybridization using non-isotopic and isotopic labeling.

A 2.3 kilobase (kb) Salmonella probe, JEO402-1, and two subfragments, F1214 (1.3 kb) and F1217 (0.8 kb), have been evaluated by colony hybridization using pure cultures of Salmonella serovars and non-salmonella bacteria. JEO402-1, and its subfragments, F1214 and F1217, hybridized to all of 156 different Salmonella serovars tested, while there was no reaction to 112 non-salmonella strains belonging to 19 genera and 37 species of Enterobacteriaceae. Together with previously published results, the JEO402-1 probe has now been shown to detect a total of 396 Salmonella strains belonging to 214 serovars of Salmonella subspecies I-VI. A total of 178 non-salmonella strains representing 23 genera and 51 species of Enterobacteriaceae have all tested negative with JEO402-1. The hybridization results obtained using a digoxigenin-labeled probe were similar to those obtained with 35S isotopic labeling when complete colony lysis was ensured.     

Cloning and characterisation of a repetitive DNA sequence fromTheileria mutans: Application as a species-specific probe

Repetitive DNA sequences were isolated from aTheileria mutans genomic library by screening withT. mutans total DNA. DNA sequence analysis demonstrated that a section of one of the clones contained a complex series of overlapping perfect repeats ranging between 99 and 20 bp in size. TheT. mutans repetitive sequence did not contain large open reading frames (ORFs), unlikeT. parva Tpr repetitive DNA sequences. When used as a hybridisation probe the repetitive sequence revealed restriction-fragment-length polymorphisms (RFLPs) between theEcoRI-digested DNAs of twoT. mutans stocks. TheT. mutans repetitive probe gave a signal with 1 ng of purifiedT. mutans piroplasm DNA and detectedT. mutans sequences in whole-blood DNA isolated from an experimentally infected animal when the piroplasm parasitaemia was equal to or above 0.4%. Oligonucleotide primers derived from the repetitive sequence allowed more sensitive detection ofT. mutans DNA by polymerase chain reaction (PCR) amplification. Using the PCR,T. mutans DNA was amplified from an experimentally infected animal with a parasitaemia of <0.1%.Nucleotide sequence data reported in this paper have been submitted to the GenBank database with accession number L16682     

Direct identification of bacterial isolates in blood cultures by using a DNA probe.

This study involved the rapid, direct identification of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Enterococcus sp., and Streptococcus agalactiae from positive blood culture bottles (BACTEC, Johnston Laboratories, Inc.) by using the AccuProbe (Gen-Probe, San Diego, Calif.) culture confirmation test. This method uses a chemiluminescent DNA probe that detects the rRNA of the target organisms. The manufacturer's instructions were modified to use a pellet of bacteria made directly from positive blood culture broth rather than a colony from an agar plate. Two separate procedures of selective centrifugation were employed in order to obtain the pellet. The first utilized a routine clinical centrifuge and a large volume of broth (10 to 12 ml) from the blood culture bottle. The second method used a microcentrifuge and less volume (1 to 1.5 ml). A total of 196 clinical specimens taken directly from positive blood culture broths were correctly identified by AccuProbe from pellets made by using the clinical centrifuge technique, while 166 clinical specimens used as negative controls failed to show hybridization. The microcentrifuge technique for obtaining pellets was performed on 105 patient specimens, and all were correctly identified. When combined with the microcentrifuge technique for pellet preparation, the AccuProbe test has several advantages: (i) direct identification of bacteria from blood culture broths, (ii) rapid turn-around time (30 min), (iii) simplicity of the procedure, and (iv) relative low cost.