Real-time PCR using fluorescent resonance emission transfer probes for HLA-B typing

HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.     

High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR)

We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. Our approach differs from previously published fluorogenic probe typing protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not specific to FRET probe analysis our system has in-built expansion capacity to 384 reactions per plate, thus making it applicable to high-throughput population screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high throughput with a low inherent error rate.     

Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes

In this study, we describe a real-time polymerase chain reaction (PCR) for genotyping all known polymorphisms of the human mannose-binding lectin 2 (MBL2) gene. These comprised two variations in the 5' regulatory region at positions -550 (H/L) and -221 (X/Y), one in the 5' untranslated sequence at position +4 (P/Q) and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B and C variants, respectively. Three reactions with two different conditions were sufficient to genotype one individual unambiguously. The three mutations in exon 1 were detected in one capillary using a sensor probe covering the three mutations, whereas amplification of the variants located upstream of the coding sequence was performed in only two reactions. Single colour detection was used for detection of the (H/L) polymorphism and multiplexing by dual colour probes was used for simultaneous genotyping of (X/Y) and (P/Q). The reliability of the system was evaluated by comparison with a conventional PCR method with sequence-specific primers (PCR-SSP). For this study, 100 individuals of Danish and 30 of African descent were analysed, and the genotypes obtained were concordant in all cases. This new method is rapid and provides reliable results without ambiguities.     

Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes

In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.     

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.

BACKGROUND: The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. OBJECTIVES: Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. STUDY DESIGN: The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. RESULTS AND CONCLUSION: Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.     

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.     

Detection and genotyping of human papillomavirus by real-time PCR assay

BackgroundDiagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients.ObjectiveOur study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples.Study designValidation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity.ResultsData showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10³ cells using in-house HPV (6, 11 and 16) viral load assays.ConclusionThe real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.     

Allele-specific in situ analysis of microchimerism by fluorescence resonance energy transfer (FRET) in nonhuman primate tissues

Rhesus monkeys are relevant models for tolerance induction. Hematopoetic chimerism is believed to be one of these strategies. The purpose of this study was to detect donor class I A locus allele specific mRNA in Rhesus monkey kidney recipient. We report here for the first time the results of frequency resonance energy transfer (FRET) hybridization technology in frozen tissues. Frequency resonance energy transfer hybridization was performed by using two Mamu-A*05 allele specific oligonucleotides: a donor probe labeled with FITC and acceptor probe conjugated to Texas Red. The PCR-SSP microchimerism analysis method produced 0.05% and 0.5% of donor DNA for Mamu-DRB1*1002 and Mamu-DRBw301/3 alleles, respectively. The donor cells were detected in mesenteric and/or inguinal lymph nodes, spleen, and liver, where the signal was the strongest. The results of FRET hybridization demonstrated the identical staining pattern in the recipient frozen tissues to that determined by PCR-SSP. Following FRET hybridization, the sections underwent immunohistochemical analysis, which revealed that donor cells had CD8+ phenotype. We demonstrate here for the first time that FRET in situ hybridization technique can be utilized for microchimerism analysis in frozen tissues. We conclude that using two donor mRNA specific oligonucleotide probes, rather than one, produce higher specificity.     

Monoclonal antibody (B27M2) subdividing HLA-B27

Because HLA-B27 shows strong association with ankylosing spondylitis, it was of interest to produce murine monoclonal antibodies to study this antigen in detail. The first such anti-B27 antibody, anti-B27M1, reacted with 100% of B27 + cells and cross-reacted with homozygous B7 cells. In the present report a second monoclonal antibody, anti-B27M2, is described which subdivides HLA-B27 into two variants. Among healthy B27⁺ individuals, 87% were B27M1⁺ and B27M2⁺, and 13% were B27M1⁺ but B27M2⁺ by standard lymphocytotoxicity assays. The specificity of B27M2 antibody for the HLA-B27 molecule was confirmed with blocking studies using F(ab′)₂ fragments of HLA alloantibodies. Both the B27M2⁺ and B27M2 variants of HLA-B27 bred true in family studies. Unlike B27M1, B27M2 antibody did not react with B7 but did react with the rare Bw47 allele.For antibody binding studies Epstein-Barr virus-transformed B lymphoblastoid cell lines were derived from normal donors KCA (by lymphocytotoxicity shown to be B27⁺, B27M2⁺. VC (B27⁺, B27M1⁺, B27M2^?), and WH (B27^?, B27M1^?, B27M2^?). Each cell line bound equivalent amounts of W6/32 (monoclonal anti-HLA-ABC): KCA and VC bound similar amounts of anti-B27M1, but only KCA bound substantial anti-B27M2 antibody. These data are consistent with a model in which all B27 antigens possess a B27M1 epitope: whereas most, but not all, possess an additional and distinct epitope, B27M2. Although the relation of these genetic variants to disease susceptibility remains to be determined, the availability of epitope-specific monoclonal antibodies should help to refine our understanding of the structure and function of HLA molecules.     

Detection and genotyping of varicella-zoster virus by TaqMan allelic discrimination real-time PCR

A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine.     

Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species

A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was done by probe hybridization and melting curve analysis, using fluorescence resonance energy transfer technology. Discrimination between Arcobacter species was straightforward, as the corresponding melting points showed significant differences with the characteristic melting temperatures of 63.5 degrees C, 58.4 degrees C, 60.6 degrees C, and 51.8 degrees C for the Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter nitrofigilis type strains, respectively. The specificity of this assay was confirmed with pure cultures of 106 Arcobacter isolates from human clinical and veterinary specimens identified by phenotypic methods and 16S rRNA gene sequencing. The assay was then used to screen 345 clinical stool samples obtained from patients with diarrhea. The assay detected A. butzleri in four of these clinical samples (1.2%). These results were confirmed by a conventional PCR method targeting the 16S rRNA gene with subsequent sequencing of the PCR product. In conclusion, this real-time assay detects and differentiates Arcobacter species in pure culture as well as in the competing microbiota of the stool matrix. The assay is economical since only one biprobe is used and multiple Arcobacter species are identified in a single test.     

Analysing the action of bacterial toxins in living cells with fluorescence resonance energy transfer (FRET)

Bacterial toxins represent small molecules produced by microorganisms. Different toxins act on specific target molecules in mammalian cells. Once discovered, bacterial toxins have been providing tools to study cellular functions and often helped the dissection of complex cellular pathways, e.g. endocytic or secretory trafficking or signal transduction, by virtue of the fact that they either block or activate their specific cellular target molecules. Purified bacterial toxins have also allowed to address many basic biological questions and have provided tools for in vitro and in vivo experimental approaches in many fields of modern biology. The understanding of how bacterial toxins act in living cells often depends on our ability to visualize the trafficking and signaling pathways of these molecules. Fluorescence microscopy and other imaging tools are essential to provide insights into the functional changes induced by these pathogens at the level of individual host cells or single target proteins. Inside a single cell we can measure and quantify the effects of bacterial toxins on specific cellular proteins by microscopic and spectroscopic techniques. Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows to study protein-protein interactions. FRET can provide distance information in the range of 3- 7 nm between fluorescently labeled bacterial proteins in the live cell and cellular target proteins expressed as chimeras with green fluorescent protein (GFP), or spectrally shifted variants thereof. The purpose of this review is to introduce readers to the main experimental setups for analyses of protein-protein interactions using FRET as well as some applications.     

具有聚集诱导发光效应的荧光探针在生物医学中的应用

荧光技术的发展为人类的科学研究提供了重要的基础.荧光材料在环境检测与保护、分子识别与示踪、生物成像以及医学诊断等领域显示出重要的应用价值,因此受到广泛重视.具有聚集诱导发光效应(aggregation induced emission,AIE)的荧光探针具有毒性低、光稳定性好、生物相容性强等优点,并且能克服传统荧光物质...     

Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with INNO-LiPA HPV genotyping extra assay

Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.     

Rapid genotyping of lipopolysaccharide-binding protein (LBP) C(1341)-->T (Leu(436)-->Phe) polymorphism by LightCycler real-time PCR

Lipopolysaccharide (LPS) is an exdotoxin found in the outer membrane of gram-negative bacteria. In circulation, LPS is bound by LPS-binding protein (LBP), which participates in cell activation by transferring LPS to CD14 and Toll-like receptor 4. A high LPS concentration may give rise to an exaggerated immune response, which may lead to septic shock during septicemia. However, LBP also neutralizes and removes LPS by transferring it to plasma lipoproteins. Recently, the presence of an amino acid-changing polymorphism in the LBP gene was reported, which, in men, was associated with sepsis and its severity and with myocardial infarction. Here, we describe a new LightCycler real-time PCR method for genotyping this LBP C(1341)-->T (Leu(436)-->Phe) polymorphism. In our study population of 393 Finnish blood donors, the genotype frequencies were: 86% TT, 13% CT and 1% CC.     

Rapid detection of the factor V Leiden (1691 G > A) and haemochromatosis (845 G > A) mutation by fluorescence resonance energy transfer (FRET) and real time PCR

A rapid method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) was used to identify the haemochromatosis genotype in 112 individuals and the factor V genotype in 134 individuals. The results were compared with conventional methods based on restriction enzyme digestion of PCR products. The two methods agreed in 244 of the 246 individuals; for the other two individuals, sequencing showed that they had been incorrectly genotyped by the standard method but correctly genotyped by FRET. The simplicity, speed, and accuracy of real time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping the haemochromatosis and factor V genes.