Hybridization Probe Set Research Articles

Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples.

The development and clinical evaluation of a LightCycler PCR assay, including an internal process control (IPC), to detect the Streptococcus pneumoniae autolysin gene in clinical samples is reported. The assay was developed to provide a second target for use in conjunction with existing pneumolysin PCR assays to increase the reliability of non-culture PCR diagnosis of pneumococcal infection. Primers amplify a 173 bp fragment of the autolysin gene (lytA), which is detected by fluorescence-labelled hybridization probes. An IPC was designed to check for the presence of PCR inhibitors and loss of assay sensitivity. The IPC product was amplified by the lytA primers and detected by a second set of hybridization probes. The analytical specificity of the autolysin PCR assay was 100% against 39 other bacterial species tested; these included related streptococci and other organisms. The assay, which could reliably detect 50 fg purified pneumococcal DNA per reaction, was capable of distinguishing between S. pneumoniae and atypical Streptococcus mitis and Streptococcus oralis strains known to contain the lytA gene. Using DNA extracts from a panel of EDTA bloods from patients with blood-culture-confirmed pneumococcal infection, the autolysin PCR had a sensitivity of 42.9%, which was similar to a previously reported TaqMan pneumolysin PCR (43.8%) run in parallel. Total agreement was shown between the autolysin assay and the pneumolysin TaqMan assay when used to test 23 culture-negative clinical samples, of which eight were positive by PCR, adding valuable clinical information. A specific autolysin-based LightCycler assay has been developed to complement pneumolysin PCR for the detection of S. pneumoniae in clinical samples. This should be a particularly useful tool for the rapid and sensitive diagnosis of pneumococcal meningitis, even after an antibiotic has been administered. However, poor sensitivity on blood samples limits its usefulness in other bacteraemic infections.

Rapid identification of commonly encountered Candida species directly from blood culture bottles.

We report a rapid-cycle, real-time PCR method for identifying six Candida spp. directly from BACTEC blood culture bottles. Target sequences in the noncoding internal transcribed spacer regions of the rRNA operon were simultaneously amplified and interrogated with fluorescent probes to identify Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae; these account for 88% of the yeast species isolated from positive blood cultures in our laboratory. Any of the first four species can be identified in a single reaction using two fluorescent hybridization probe sets. The antifungal-resistant species C. krusei and C. lusitaniae are detected in a second reaction, also with two probe sets. The assay was validated with DNA extracted from BACTEC blood culture bottles positive for yeasts (n = 62) and was 100% concordant with culture identification based on biochemical and morphological features of C. albicans (n = 22), C. parapsilosis (n = 10), C. tropicalis (n = 1) C. glabrata (n = 22), C. krusei (n = 2), and C. lusitaniae (n = 1). No cross-reactivity was observed in blood culture samples growing yeasts other than the above-mentioned species (n = 4), in those growing bacteria (n = 12), or in the absence of microbial growth. Our assay allows rapid (</=2 h) and specific detection of the most common Candida spp. directly from positive blood cultures and may facilitate delivery of optimal antifungal therapy.

Detection of Neisseria Meningitidis in clinical samples by a duplex real-time PCR targeting the porA and ctrA genes.

In recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused by Neisseria meningitidis. We developed and evaluated a N. meningitidis LightCycler real-time duplex PCR (NM-LCdPCR) capable of simultaneously detecting and distinguishing between two separate genes on the N. meningitidis genome. The NM-LCdPCR was developed on the LightCycler platform (Roche Diagnostics, Castle Hill, NSW, Australia) and comprised two primer pairs and two hybridization probe sets, enabling the detection of both the porA and ctrA genes within the same reaction mix. To distinguish between the fluorescence emitted by each hybridization probe set, each downstream probe was labeled with a different fluorophore (either LC-Red640 or LC-Red705). The results obtained by the NM-LCdPCR were then compared with the results obtained by a mono-specific LightCycler assay targeting the porA gene only (porA-LCPCR). One-hundred and forty-eight clinical samples from patients with suspected meningococcal infection were evaluated. The results of the NM-LCdPCR and porA-LCPCR gave 100% agreement; N. meningitidis DNA was detected in 25 samples whereas 123 samples were negative by both assays. The breakdown of the NM-LCdPCR results show that both genes were detected in 26 of the 28 positive samples. By targeting two separate N. meningitidis genes, the NM-LCdPCR has the potential to prevent the false-positive results which may arise from sequence variation. In addition, the ability to detect and discriminate between the two different N. meningitidis genes within the same reaction mix offers a rapid means for confirming the presence of N. meningitidis DNA in clinical samples, thereby reducing the need for subsequent confirmatory assays to be performed. The sensitivity and specificity of the NM-LCdPCR assay, combined with its ability to detect and discriminate both the N. meningitidis porA and ctrA genes, make it suitable for the diagnosis of N. meningitidis infections in the routine clinical laboratory.

Multitarget Fluorescence In Situ Hybridization Assay Detects Transitional Cell Carcinoma in the Majority of Patients with Bladder Cancer and Atypical or Negative Urine Cytology

The multitarget fluorescence in situ hybridization (FISH) probe set UroVysion (Vysis, Downers Grove, Illinois), containing probes to chromosomes 3, 7 and 17, and to the 9p21 band, has been recently shown to have high sensitivity and specificity for detecting transitional cell carcinoma. In this study we retrospectively tested 120 urine samples from patients with atypical, suspicious and negative cytology for whom concurrent and followup bladder biopsy data were available. We evaluated the ability of FISH to identify malignant cells in cytologically equivocal or negative cases. Archived slides from 120 voided (47) or instrumented (73) urine cytology specimens from patients with concurrent bladder biopsy and a minimum of 12 months of biopsy followup were subjected to hybridization with UroVysion. The cohort included patients with biopsy proven transitional cell carcinoma, which was grades 1 to 3 in 23, 35 and 24, respectively, and stages pTis in 3, pTa in 64, pT1 in 6, pT2 in 6 and pT4 in 3, while it showed negative histology in 38. Cytology findings were suspicious, atypical and negative for transitional cell carcinoma in 31, 49 and 40 cases, respectively. A positive FISH result was defined as 5 transitional cells or greater with a gain of 2 or more of chromosomes 3, 7 or 17, 12 cells or greater with 9p21 deletion, or 10% or greater of cells with isolated trisomy of 1 of chromosomes 3, 7 and 17. All except 12 of the 82 biopsy proven transitional cell carcinoma cases (11 pTa and 1 pT1 tumors) were positive by FISH (85% sensitivity). Sensitivity in patients with suspicious, atypical and negative cytology was 100%, 89% and 60%, respectively. Nine patients with atypical cytology had positive FISH in the setting of a negative concurrent bladder biopsy. However, 8 of these 9 patients (89%) had biopsy proven transitional cell carcinoma within 12 months following the date when the sample tested by FISH was obtained. The last of these patients with false-positive results had previously documented pTis disease, which was also present in the next bladder biopsy 15 months following the positive FISH result. The remaining 29 specimens from patients with negative biopsy and a negative 12-month followup tested negative by FISH (97% overall specificity). The UroVysion FISH assay provides high sensitivity and specificity to detect transitional cell carcinoma in cytologically equivocal and negative urine samples. These results emphasize the important role of this assay in the management of bladder cancer.

Fluorescence In Situ Hybridization as an Ancillary Tool in Urine Cytology to Diagnose Urothelial Carcinoma

Urothelial carcinoma represents a diagnostic challenge for cytopathologists, especially for diagnosing low-grade tumors. The poor sensitivity of urine cytology makes it difficult for the pathologist to identify these tumors. In the past few years, researchers have sought more sensitive and specific methods, and the focus has been on identifying chromosomal abnormalities in tumor cells. A multicolor, multitarget, interphase fluorescence in situ hybridization (FISH) probe set containing probes to the centromeres of chromosomes 3, 7, 17, and 9p21 band has been shown to be more sensitive and specific for detecting urothelial carcinoma in urine specimens when compared with urine cytology. This is helpful in cases in which the urine cytology is equivocal. Early diagnosis can significantly reduce the morbidity and mortality of urothelial carcinoma. FISH can also be used for monitoring patients with noninvasive urothelial carcinoma for tumor recurrence.

Phylogenetic analysis of methanogens from the bovine rumen.

Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified. Total DNA from clarified bovine rumen fluid was amplified using primers specific for Archaeal 16S rRNA gene sequences (rDNA). Phylogenetic analysis of 41 rDNA sequences identified three clusters of methanogens. The largest cluster contained two distinct subclusters with rDNA sequences similar to Methanobrevibacter ruminantium 16S rDNA. A second cluster contained sequences related to 16S rDNA from Methanosphaera stadtmanae, an organism not previously described in the rumen. The third cluster contained rDNA sequences that may form a novel group of rumen methanogens. The current set of 16S rRNA hybridization probes targeting methanogenic Archaea does not cover the phylogenetic diversity present in the rumen and possibly other gastro-intestinal tract environments. New probes and quantitative PCR assays are needed to determine the distribution of the newly identified methanogen clusters in rumen microbial communities.

Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 10(1) to 10(7) HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

A rapid and sensitive approach to mutation detection using real-time polymerase chain reaction and melting curve analyses, using BRCA1 as an example

The detection of 2 recurrent mutations in the BRCA1 gene (Ashkenazi Jewish and Dutch populations) was studied with real-time polymerase chain reaction (PCR) and melting curve analyses. PCR products were designed around the 185delAG in exon 2 and the single- base substitution 2841G>T in exon 11. Hybridization probe sets were designed for both PCR products, with each probe overlapping the specific mutation. The exon 11 probe set also covered another mutation, the 2814insA. The 39 end of the 59 probe was labeled with fluorescein isothiocyanate and the 59 end of the 39 probe with LightCycler Red 640 (Roche Diagnostics, Indianapolis, IN). The 185del and 2841G mutations were easily detected with the hybridization probes, resulting in dual peaks for heterozygotes in melting curve analyses. The differences in melting characteristics of the heteroduplexes in heterozygotes were not detectable with SYBR Green I. For known mutations, melting curve analyses using hybridization-specific probes provide a sensitive, rapid, and efficient approach to mutation detection.

Expression of mouse Tla region class I genes in tissues enriched for ?? cells

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to γδ T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for γδ T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain γδ T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing γδ T cells further extends the list of potential candidates for antigen presentation to γδ T cells.

Expression of mouse Tla region class I genes in tissues enriched for gamma delta cells.

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to gamma delta T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for gamma delta T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent and transcripts from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain gamma delta T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing gamma delta T cells further extends the list of potential candidates for antigen presentation to gamma delta T cells.

Biogenesis of mitochondria: Mapping of transcripts from the oli2 region of mitochondrial DNA in two grande strains of Saccharomyces cerevisiae

A series of 14 contiguous restriction fragments of yeast mitochondrial DNA that cover a 5.2 kb segment including the oli2 gene provided a set of hybridization probes that were used to analyze gel fractionated mitochondrial RNA immobilized on diazotized paper. Of the nine oli2 region transcripts in grande strain J69-1B (class I), the five largest (5,100, 4,500 (most prominent), 3,900, 3,600 and 3,000 nucleotides) contain the oli2 coding sequence; all appear to have the same 3' end. The four smallest J69-1B transcripts (1,900, 1,700, 800 and 600 nucleotides) contain only sequences down-stream of the oli2 gene. A 1.8 kb DNA region specifying these four transcripts is deleted from grande strain JM6 (class 11); these transcripts are thus dispensible for respiratory function. The five oli2 transcripts of JM6 (3,000, 2,400 (most prominent), 2,000, 1,700 and 1,150 nucleotides) all contain the oli2 coding sequence and are each related to a corresponding J69-1B transcript; the 5' and 3' ends match in each case, but the JM6 transcripts are about 2,000 nucleotides shorter. In particular, the putative oli2 mRNA has a 5' leader of about 1,400 nucleotides, a coding region of 780 nucleotides, and a variant 3' tail that is about 2,300 nucleotides in J69-1 B, and about 200 nucleotides in JM6.