Resonance Energy Transfer Hybridization Probes Research Articles

Real-Time PCR Method for Detection of Zygomycetes

Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/mul, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

BackgroundA simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated.MethodsConsensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria.ResultsBlood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample.ConclusionThis real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

Real-time PCRs for detection of Trichomonas vaginalis β-tubulin and 18S rRNA genes in female genital specimens

Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. The incidence of trichomoniasis in developed countries has decreased substantially during the past decade, but high prevalence of this disease can still be found in rural and remote areas of Australia. Clinical manifestations of symptomatic women are generally non-specific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. Current diagnosis of T. vaginalis relies on the visualization of motile organisms in a wet-mount preparation. Culture is used mainly in reference laboratories. The latter two methods require viable organisms and would not be suitable for use where transportation of specimens can be delayed. Two real-time fluorescence resonance energy transfer (FRET) hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the beta-tubulin and 18S rRNA genes. We tested 500 randomly selected female patients, in an STD setting, for T. vaginalis DNA. The FRET PCRs targeting the beta-tubulin gene and the 18S rRNA gene detected 96 % (85/89) and 100 % (89/89) , respectively, of the positive specimens (first-void urine sample or genital swabs). Wet-mount microscopy was performed on 76 of these PCR-positive specimens and showed a sensitivity of 38 % (29/76). The prevalence, by PCR, of trichomoniasis was 18 % in this study. The two real-time PCRs developed in this study, targeting different genetic regions of the organism, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.

Direct Molecular Haplotyping of the IVS-8 Poly(TG) and PolyT Repeat Tracts in the Cystic Fibrosis Gene by Melting Curve Analysis of Hybridization Probes

Molecular haplotyping is a developing technology with great potential for use in clinical diagnostics. We describe a haplotyping method that uses PCR combined with hybridization probes. We designed a LightCycler assay that uses fluorescence resonance energy transfer hybridization probes to haplotype the poly(TG) and polyT (TG-T) tract in the IVS-8 region of the CFTR gene. The reporter probe was designed as a perfect match to the TG12-5T allele. Analysis of 132 samples revealed 9 unique derivative melting temperatures (Tms); the lowest was 42.4 degrees C and the highest was 63.6 degrees C. The lowest Tms were in the TGn-9T group, the intermediate Tms in the TGn-7T group, and the highest Tms in the TGn-5T group. Haplotype frequencies were highest (39%) for TG11-7T and lowest (0.4%) for TG13-5T. Different combinations of polymorphisms under the reporter hybridization probe had unique and characteristic Tms. This property enables genotyping as well as determination of the phase of multiple variants under the probe, a principle we demonstrated by haplotyping the TG-T repeat tract in the IVS-8 region of the CFTR gene.

Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction

A multiplex real-time polymerase chain reaction (RT-PCR) targeting the mecA and nuc genes was developed for the detection of methicillin resistance and identification of Staphylococcus aureus. Novel mecA and nuc primers and fluorescence resonance energy transfer hybridization probes specific for the mecA and nuc genes were evaluated. The assay was performed using the LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) and evaluated against the traditional gel-based multiplex PCR (PCR-gel) method currently used at Royal Perth Hospital. Clinical isolates ( n = 222) and isolates from a culture collection library ( n = 206) were tested by both assays in parallel. The RT-PCR assay was 100% sensitive and specific for the detection of methicillin resistance and for the identification of S. aureus when compared with the PCR-gel assay. Results from the RT-PCR assay showed 5 isolates with lower efficiency fluorescence curves for the nuc gene PCR fragment. DNA sequencing showed mutations within the region of the probe-binding sites compared with the reference strain. The results of the RT-PCR assay were available within 2 h. This rapid mecA/ nuc RT-PCR assay is a suitable and practical tool for the routine detection of methicillin resistance and identification of S. aureus, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow.

Use of fluorescence resonance energy transfer hybridization probes to evaluate quantitative real-time PCR for diagnosis of ocular toxoplasmosis.

Toxoplasma gondii infection is an important cause of chorioretinitis in Europe and the United States. Ophthalmological examination and a good clinical response to adequate therapy mainly support ocular toxoplasmosis diagnosis. However, clinical diagnostic may be difficult in some atypical cases. In these cases, laboratory confirmation, based on detection of local specific antibodies and parasite DNA by conventional PCR, is therefore important to confirm the disease etiology. More recently, real-time PCR has been developed to improve prenatal congenital toxoplasmosis diagnosis. We therefore examined the diagnostic value of quantitative real-time PCR for the detection of T. gondii in aqueous humor samples, associated with quantification of human beta-globin to control sample quantitative quality, by using a double fluorescence resonance energy transfer hybridization probes system with a double fluorescence reading. Of the 23 the clinically toxoplasmosis suspect patients, 22 showed serological evidence of exposure to Toxoplasma; one had a serological profile indicative of active infection. The analysis of paired aqueous humor and serum samples revealed an intraocular antibody production in 9 of 23 cases (39.1%). The quantitative real-time PCR revealed positive and high parasite numbers and high Toxoplasma/human genome ratios in three cases. Furthermore, PCR was the only positive confirmatory test in two cases (11.1%). None of the patients included in the control group (n = 7) had evidence of either local specific antibody production or T. gondii DNA detection, suggesting a good relative assay specificity. On the whole, quantitative real-time PCR appears to be useful for diagnosing atypical ocular toxoplasmosis presentations.

Combined factor V leiden (G1691A) and prothrombin (G20210A) genotyping by multiplex real-time polymerase chain reaction using fluorescent resonance energy transfer hybridization probes on the Rotor-Gene 2000.

Several methods have been developed to detect common single point mutations in the factor V and prothrobin genes that are risk factors for thrombophilia. Most are based on PCR followed by restriction enzyme digestion and electrophoresis (RFLP), but gel analysis has certain limitations, and alternative detection methods, including real-time PCR, have therefore been developed. In this study we developed and evaluated a combined factor V Leiden and prothrombin (G20210A) genotyping method based on multiplex real-time PCR with fluorescent resonance energy transfer (FRET) hybridization probes on the Rotor-Gene 2000. Two hundred subjects were screened for the two mutations. The FRET assay clearly discriminated among wild-type, homozygous and heterozygous status for the two mutations, and the results were in full agreement with those of the RFLP assay. This robust FRET probe-based assay also has a higher throughput capacity than conventional methods, handling up to 72 samples in 90 min.

Parasite load in guinea pig foetus with real time PCR after maternofoetal transmission of Toxoplasma gondii.

Parasite loads of different tissues were assessed in guinea pig foetus after maternal infection. Twelve female guinea pigs were infected with 100 cysts of the 76 K strain of Toxoplasma gondii by the oral route. Inoculation was performed 20 +/- 5 days (G20) or 40 +/- 5 days (G40) after the beginning of gestation. Gestational age was determined by progesterone assay. Maternal and foetal organ samples were taken 60 days after the beginning of gestation. Parasite loads (from placenta, amniotic fluid (AF), cord blood (CB), foetal brain, liver, lung and spleen) were assessed by a real-time PCR quantification using fluorescence resonance energy transfer (FRET) hybridization probes on the Light Cycler. Congenital transmission was proven by the presence of parasites in blood or tissue samples of the foetus in 84.6% (11/13) and 100% (16/16) of cases after inoculation on G20 and G40, respectively. The quantitative analysis of our results after inoculation at G20 and G40 has allowed us to determinate the positive parasitic loads as a function of the origin of the sample and the period of inoculation. The parasite loads expressed as log (parasite/g) were low in AF and CB samples: 1.49 +/- 0.50 and 1.05 +/- 0.10 at G20 and 1.21 +/- 0.36 and 1.20 +/- 0.42 at G40 respectively. In contrast the placenta and the different foetal tissues had higher parasite burdens: 2.89 +/- 0.54 to 5.30 +/- 0.51 at G20 and 2.81 +/- 0.71 to 3.65 +/- 0.59 at G40. All the placentae were positive for parasites even in the two cases with no proven transmission. Real time quantitative PCR using the hybridization probe was a very sensitive and reproducible technique to study the kinetics of congenital toxoplasmosis in the guinea pig model wich is close to that of humans.

Real-time PCR for the rapid detection of vanA and vanB genes

A real-time PCR assay suitable for use on the Roche LightCycler platform was developed to replace an existing gel-based PCR assay for the simultaneous detection of the vanA & vanB genes in enterococcal isolates. Novel Fluorescence Resonance Energy Transfer (FRET) hybridization probes were designed. The multiplex real-time PCR assay and the existing gel-based assay were 100% concordant and both correctly detected the vanA or vanB genes in 4/4 VanA E. faecium and 25/25 VanB E. faecium. Additionally, 1/1 VanC1 E. gallinarum, 1/1 VanC2 E. casseliflavus and 47/47 vancomycin susceptible enterococci were negative for the vanA and vanB genes in both PCR assays. Results were available within 1.5 h for the real-time PCR assay compared to up to 5.5 h for the conventional PCR assay.

Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes.

The diagnosis of congenital toxoplasmosis frequently relies on PCR tests of amniotic fluid (AF). A duplex real-time quantitative PCR test based on fluorescence resonance energy transfer was developed to quantify the parasite load and to decrease the risk of contamination. An internal control based on the detection of 10 pg mouse DNA added to the AF was included to check for PCR efficiency. The relationship between the parasite load and the occurrence of ultrasonographic abnormalities in 87 samples of AF was analyzed. Seven AF (8%) had a parasitic load > 10(3); 14 (16%) had > 10(2)-< or =10(3); 26 (30%) had > 10-< or = 10(2); and 40 (46%) had < or = 10 parasites/ml. Four of the six AF with cerebral ventriculomegaly had >10(3) parasites/ml. The other two had 130 and 24 parasites/ml, respectively. No parasitic loads of > 10(3) parasites/ml and no ultrasonographic abnormalities were observed in the 11 AF with maternal toxoplasmosis in the third trimester. Therefore, there is a trend to associate high parasite count with ultrasonographic abnormality, but the main concern remains early maternal infection. The importance of quantification should be better evaluated with postnatal studies. The duplex LightCycler PCR test currently provides rapid and safe results.

Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler.

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).

Real-Time PCR for Diagnosis and Follow-Up of Toxoplasma Reactivation after Allogeneic Stem Cell Transplantation Using Fluorescence Resonance Energy Transfer Hybridization Probes

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify Toxoplasma gondii DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 x 10(6) to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to </=0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.