PCR amplification and phylogenetic analysis of groESL operon sequences from Ehrlichia ewingii and Ehrlichia muris

Broad-range PCR primers were used to amplify part of the groESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.     

Determination of the nucleotide sequences of heat shock operon groESL and the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys for phylogenetic and diagnostic studies

The 1,670-bp nucleotide sequence of the heat shock operon groESL and the 1,236-bp sequence of the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys were determined. The topology of the groEL- and gltA-based phylogenetic tree was similar to that derived from 16S rRNA gene analyses with distances. Both groESL- and gltA-based PCRs specific to A. platys were also developed based upon the alignment data.     

Comparison of the nucleotide sequences of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from Northern California

We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes. Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444 Ep-ank gene of the HGE agent and E. equi isolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.     

Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR.

A two-step PCR method for diagnosis of canine infectious cyclic thrombocytopenia was established. Three primers derived from the 16S and rRNA gene sequence were used to amplify genomic DNA specifically from Ehrlichia platys. Two-round amplification with DNA templates prepared from E. platys-infected blood specimens produced 742- and 385-bp fragments, but these products were not found when an Ehrlichia canis-infected blood sample and Escherichia coli were used. This method, for which the minimum detectable copy number in the blood specimen was estimated to be five ehrlichia inclusion within platelets, is more sensitive than single PCR amplification. These results demonstrate that this two-step PCR is highly sensitive and efficient for detecting the etiologic agent of canine infectious cyclic thrombocytopenia in blood. The same technique was applied to blood specimens collected from a dog inoculated with E. platys. Amplification of the target DNA fragments was observed with blood collected on the fifth day after inoculation, which indicates that this method is also feasible for early diagnosis of E. platys infection.     

Molecular analysis of the Haemophilus ducreyi groE heat shock operon.

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L. M. Parsons, M. Shayegani, A. L. Waring, and L. H. Bopp, J. Clin. Microbiol. 27:1441-1445, 1989). In the present study, DNA sequencing of one of the diagnostic probes revealed two adjacent open reading frames (ORFs). These H. ducreyi ORFs and the encoded proteins show significant homology with the groE genes and GroES and GroEL heat shock proteins from several bacterial pathogens and with conserved eukaryotic 60-kDa heat shock proteins. The first H. ducreyi ORF (groES) is preceded by sequences similar to those of the Escherichia coli consensus heat shock promoters and is 288 nucleotides long and is capable of encoding a protein of 10.3 kDa. The second ORF (groEL) is 1,641 nucleotides long and is capable of encoding a protein of 57.8 kDa. Northern (RNA blot) analysis demonstrated the presence of a high level of groE mRNA in exponential-phase H. ducreyi grown in hemin broth at the organism's optimal growth temperature (33 degrees C), with increased levels seen following heat shock. Heat shock also increased the thermostability of the organisms, since stressed cells were more resistant to the lethal effects of rapid chilling. Electrophoretic analysis and immunoblots demonstrated that the predominant protein produced by exponential-phase H. ducreyi was a heat-inducible, immunoreactive protein of approximately 60 kDa (GroEL). Also, H. ducreyi groE mRNA and GroEL were expressed and inducible by heat in E. coli. This is the first report describing the cloning, sequencing, and expression of H. ducreyi protein-encoding genes.     

Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells

A 60-kilodalton (kDa) immunodominant antigen of Legionella pneumophila is a heat shock protein (HSP) of the GroEL class of HSPs. The gene (htpB) coding the 60-kDa protein was localized to a 3.2-kilobase DNA fragment of L. pneumophila cloned into pUC19 (pSH16) (P. S. Hoffman, C. A. Butler, and F. D. Quinn, Infect. Immun. 57:1731-1739, 1989). The nucleotide sequence of the DNA fragment cloned into M13 confirmed two open reading frames, htpA and htpB, that code for proteins of 96 and 548 amino acids, respectively. A consensus heat shock promoter sequence upstream of the start of htpA was identified, and no obvious promoter sequences were detected upstream of htpB. Amino acid sequence comparison studies revealed that the L. pneumophila HtpB protein exhibited 76% homology with the 65-kDa protein of Mycobacterium tuberculosis and 85% homology with both GroEL of Escherichia coli and HtpB of Coxiella burnetii. A comparison of the amino acid sequences among these proteins revealed several regions of nearly absolute sequence conservation, with the variable regions occurring in common areas. The purified L. pneumophila 60-kDa protein was antigenic for human T lymphocytes. Indirect fluorescent antibody studies indicated that the 60-kDa protein may be located in the periplasm or expressed on the surface by intracellular bacteria, suggesting that a stress-related mechanism may be involved in the expression of this immunodominant antigen.     

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences.

The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no "shadow bands". These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations.     

PCR amplification of HIV-1 proteinase sequences directly from lab isolates allows determination of five conserved domains.

HIV-1 replication requires limited proteolysis of gag and gag-pol encoded precursor proteins by a specific viral proteinase (PR). Sequences of 20 different HIV-1 strains were compared in order to determine regions of conservation and variability within the PR gene. Viral strains included: (a) five new ones derived from New Orleans patient isolates, (b) four established ones grown in our laboratory, (c) eight, whose sequences were published in the Los Alamos Data Base (1990), (d) one Ugandan, and (e) two Brazilian isolates. In the first two groups, HIV proviral DNA extracted from infected lymphocytes was grown in tissue culture and directly amplified by PCR using specific primers flanking the PR gene. Amplified DNA was directly sequenced using a modified di-deoxy sequencing procedure. Sequence data showed a 25% variation among the 20 different HIV strains studied at the amino acid level, including 8% nonconservative changes and 17% conservative changes. Moreover, five noncontiguous regions were able to be delineated in which the PR showed no amino acid changes. These areas included amino acids (I) 1-9 (amino terminal sequence); (II) 21-32 (sequence around the active site); (III) 47-56 (top of the flap); (IV) 78-88; and (V) 94-99 (carboxy terminal sequence). Our results are consistent with those obtained from X-ray crystallography studies as well as single site mutational analysis.     

PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria.

A total of 129 reference and clinical strains of rapidly growing mycobacteria (RGM) belonging to 10 taxonomic groups were studied for restriction fragment length polymorphism patterns from a PCR-amplified 439-bp segment of the 65-kDa heat shock protein (HSP) gene. Of 24 endonucleases evaluated, restriction fragment length polymorphism patterns produced by HaeIII and BstEII and then by AciI and CfoI gave the best separation. Sixty percent of all RGM taxa studied were differentiated by HaeIII digests alone. Single unique patterns were observed with HaeIII and/or BstEII for Mycobacterium fortuitum (100%), M. chelonae (94%), M. abscessus (96%), M. smegmatis (100%), M. mucogenicum (formerly the M. chelonae-like organism) (100%), and the sorbitol-negative third biovariant of M. fortuitum (100%). Evidence is presented in support of two subgroups within M. peregrinum, M. smegmatis, and the unnamed third biovariant of M. fortuitum (sorbitol positive and sorbitol negative). PCR-based technology provides a rapid, accurate system for the identification of clinically important species of RGM which should be particularly useful for reference laboratories.     

PCR amplification of DNA from stained cytological smears.

DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.     

Detection of Ehrlichia risticii from feces of infected horses by immunomagnetic separation and PCR.

Potomac horse fever, caused by Ehrlichia risticii, is an important disease of equines. The major features of the disease are fever, leukopenia, and diarrhea. The organism has been detected from the blood mononuclear cells of infected horses, but its presence in the feces has not been known. A method for immunomagnetic separation of E. risticii from the feces of infected horses was developed, and the separated organisms were detected by PCR. Coating immunomagnetic beads (Dynabeads) with a 1:5 dilution of rabbit anti-E. risticii serum and incubating the Dynabeads with fecal samples for 25 min at room temperature gave optimum results. E. risticii was detected from the feces during the course of diarrhea from two experimentally infected horses. In horse 1, watery diarrhea occurred from days 11 to 16 postinfection (p.i.), after which the feces became soft on day 17 p.i. and then returned to normal. The organisms were first detected from the feces on day 11 p.i., peaked on day 13 p.i., and then gradually decreased until day 16 p.i., after which they became undetectable. In horse 2, first, on day 12 p.i., there was soft feces which continued and progressed to diarrhea on day 17 p.i. The feces became normal after day 18 p.i. The organisms in the feces of this horse were first detected on day 12 p.i. and peaked on day 14 p.i., after which they declined until day 16 p.i. and then became undetectable. In both horses, the number of organisms in the mononuclear cells peaked on days 10 and 11 p.i., respectively, 3 days prior to the respective peaks in the feces. E. risticii was not detected from the plasma samples obtained from these horses. There was a drastic reduction in PCR amplification of E. risticii DNA for fecal samples stored frozen at -20 degrees C in comparison with those stored at 4 degrees C. The presence of the organism in the feces only during the soft- or diarrheal-feces phase supports the previous hypothesis that the diarrhea is caused by the organisms replicating in cells lining the intestines. This rapid simple method of detection of the organisms from the feces will be helpful in diagnostic and epidemiologic studies of Potomac horse fever.     

A novel regucalcin gene promoter region-related protein: comparison of nucleotide and amino acid sequences in vertebrate species

The molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein (RGPR-p117) from bovine, rabbit and chicken livers was investigated using rapid amplification of cDNA endo (RACE) method. Their nucleotide and amino acid sequences were compared with human, rat and mouse sequences published previously. RGPR-p117 of bovine, rabbit and chicken livers consisted of 1052, 1045, and 929 amino acid residues with calculated molecular mass of 117, 114, and 103 kDa, and estimated pI of 5.64, 5.84, and 5.59, respectively. Comparison analysis revealed that the nucleotide sequences of RGPR-p117 from mammalian species were highly-conserved in their coding region, and the homologies were at least 72.9%. The RGPR-p117 proteins in mammalian species consisted of 1045-1060 amino acids, and had 63.1-90.2% identity. Meanwhile, the nucleotide and amino acid sequences of chicken RGPR-p117 had at least 36.4 and 43.7% identities, respectively. Phylogenetic analysis showed that RGPR-p117 in six vertebrates appears to form a single cluster. Mammalian RGPR-p117 conserved a leucine zipper motif. Moreover, the analysis for subcellular localization of RGPR-p117 from six vertebrates showed the probability of nuclear localization >52.2%; the nuclear localization in rat and mouse was 78.3%. This study demonstrates a great conservation of RGPR-p117 genes throughout evolution.     

PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

The polymerase chain reaction (PCR) DNA amplification method is a powerful new tool for the retrospective analysis of paraffin-embedded tissue (PET). The technique has afforded the sensitive and specific detection of nucleic acid sequences associated with genetic and infectious diseases. However, PET processing conditions vary in their suitability for amplification. The authors have examined the effects of 11 fixatives at three fixation times. The effect of fixation was measured by the ability of the DNA in a treated tissue to serve as a template for the amplification of DNA fragments that ranged from 110 to 1,327 base pairs in length. Specimens fixed in acetone or 10% buffered neutral formalin were found to be best suited for subsequent analysis by PCR. A second group of fixatives, including Zamboni's, Clarke's, paraformaldehyde, formalin-alcohol-acetic acid, and methacarn, compromised amplification efficiency. Tissues treated with Carnoy's, Zenker's, or Bouin's, respectively, were even less desirable for amplification analysis.     

Comparison of methods for extraction of nucleic acid from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences.

The sensitivity of PCR for the amplification of target nucleic acid sequences in clinical diagnostics may often be reduced due to the presence of inhibitory factors. Hemolytic serum contains a number of PCR inhibitors, one of which is hemin. In this study we have found that conventional methods of DNA extraction were not sufficient for the removal of PCR-inhibitory compounds in hemolytic serum. We have therefore compared the efficiency of several commercial and noncommercial methods of nucleic acid purification from hemolytic serum samples prior to PCR amplification. Separation with the QIAamp HCV kit, dialysis with Millipore filters, and bovine serum albumin absorption were all found to be suitable extraction methods for eliminating inhibitors from hemolytic serum for PCR amplification. Using these methods we were able to detect very low levels of hepatitis B virus DNA in hemolytic serum.     

PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.

RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary.     

Identification of Ehrlichia chaffeensis by nested PCR in ticks from Southern China

A total of 717 ticks collected from southern China were examined by nested PCR for the presence of Ehrlichia chaffeensis. Sixteen (55. 2%) of 29 adult Amblyomma testudinarium ticks and 28 (11.7%) of 240 adult and at least 4.2% of 215 nymphal (pooled specimens) Haemaphysalis yeni ticks tested positive. Four other species of ticks were negative. Selected positive amplicons were confirmed by DNA sequencing.     

Specific amplification of Rickettsia japonica DNA from clinical specimens by PCR.

The gene encoding the 17,000-molecular-weight genus-common antigen (17K genus-common antigen) has been cloned and sequenced from Rickettsia japonica. The primer pair used for PCR was designed from this sequence. A 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and also the DNA from blood clots of patients with spotted fever group rickettsiosis. The results indicated that this method is suitable for the diagnosis of spotted fever group rickettsiosis in Japan.