HLA-B27 genotyping by Fluorescent Resonance Emission Transfer (FRET) probes in real-time PCR

Faner, Rosa; Casamitjana, Natàlia; Colobran, Roger; Pujol-Borrell, Ricardo; Palou, Eduard; Juan, Manel

doi:10.1016/j.humimm.2004.05.009

Cited by 20 publications

(10 citation statements)

References 25 publications

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“…FRET probes provide information on the polymorphisms via hybridization of the sensor probe to the DNA strand. To do so, an analysis of the generation of a melting curve is required. The slow denaturalization of the probes, once they are hybridized to the amplimer, reveals which amplimers contain mismatches in relation with the sensor probe sequence (lower Tms) and which contain the full complementary sequence to the sensor probe (higher Tm) .…”

Section: Methodsmentioning

confidence: 99%

“…FRET probes provide information on the polymorphisms via hybridization of the sensor probe to the DNA strand. To do so, an analysis of the generation of a melting curve (16,18) is required. The slow denaturalization of the probes, once they are Table 1 Primers and probes used for HLA-DQ2/DQ8 genotyping and HLA-DQB1*02 homozygosity analysis HLA-DQ2/DQ8 genotyping Primers/probes…”

Section: Hla-dq2/dq8 Genotyping With Light Cycler Hybridization Fluormentioning

confidence: 99%

“…hybridized to the amplimer, reveals which amplimers contain mismatches in relation with the sensor probe sequence (lower Tms) and which contain the full complementary sequence to the sensor probe (higher Tm) (16,18).…”

Section: Hla-dq2/dq8 Genotyping With Light Cycler Hybridization Fluormentioning

confidence: 99%

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HLA‐DQ2/DQ8 and HLA‐DQB102* homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

et al. 2014

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Celiac disease (CD) is a complex autoimmune disorder caused by ingestion of gluten in genetically susceptible individuals. Different genetic risk factors have been identified, but virtually all patients are human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8 positive. We describe a new, fast, accurate and simple real-time polymerase chain reaction (PCR)-based assay for the genotyping and homozygosity analysis of the CD-related HLA alleles. The assay overcomes the major limitations of protocols currently in use, allowing HLA-DQ2/DQ8 genotyping by using only three real-time PCR reactions. For the appraisal of DQ2 homozygosity, only one more reaction is needed. These reactions are easily automated and suitable for large screening studies in diagnostic procedures, as it is demonstrated by their successful application in our HLA diagnostic laboratory. Finally, we assessed the clinical relevance of this real-time PCR-based assay by studying a cohort of fully characterized patients. As expected, all CD patients had at least one of the CD-associated alleles, and the highest CD risk was indicated by the presence of the HLA-DQ2.5 heterodimer (HLA-DQA1*05-DQB1*02) with HLA-DQB1*02 in homozygosity.

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Section: Methodsmentioning

confidence: 99%

Section: Hla-dq2/dq8 Genotyping With Light Cycler Hybridization Fluormentioning

confidence: 99%

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HLA‐DQ2/DQ8 and HLA‐DQB102* homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

et al. 2014

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“…Typically, this method is performed only by specialized laboratories. Alternative methods for HLA‐B27 typing include (i) HLA‐B27 genotyping by polymerase chain reaction (PCR) assays based on sequence‐specific primers (PCR‐SSP; Olerup, ), (ii) HLA‐B27 genotyping by PCR assays based on sequence‐specific oligonucleotides (PCR‐SSO; Dominguez et al, ), (iii) HLA‐B27 genotyping by fluorescent resonance emission transfer (FRET) probes in real‐time PCR (Faner et al, ), (iv) HLA‐B27 genotyping by SNP analysis using TaqMan technology (Behrens and Lange, ), (v) HLA‐B27 genotyping by an allele‐specific PCR melting assay (Seipp et al, ), and (vi) enzyme‐linked immunosorbent assays (Chou et al, ). Again, these techniques require specialized knowledge and extensive quality‐control procedures, and therefore are performed mainly by specialized tissue‐typing laboratories.…”

Section: Commentarymentioning

confidence: 99%

Flow Cytometric Screening for the HLA‐B27 Antigen on Peripheral Blood Lymphocytes

et al. 2005

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HLA‐B27 is common to the entire group of seronegative spondyloarthropathies, and assessment of HLA‐B27 is of diagnostic importance if any of these diseases are considered. Among the alternatives to traditional typing by the complement‐dependent cytotoxicity assay, flow cytometric HLA‐B27 screening is most widely used in general diagnostic laboratories, as it is simple, rapid, and cost effective. This unit describes screening for HLA‐B27 on peripheral blood lymphocytes using more than one HLA‐B27 monoclonal antibody to detect possible cross‐reactivity with non‐HLA‐B27 antigens. Screening is hampered by the lack of true monospecific anti‐HLA‐B27 monoclonal antibodies. Cross‐reactivities of anti‐HLA‐B27 with other HLA‐B antigens have been reported. The authors recommend use of GS145.2 and FD705 antibodies, as this combination avoids most false‐positive conclusions. Lyse‐and‐wash sample processing is employed. A multiparameter flow methodology is applied. Three to four parameters–forward scatter, side scatter, HLA‐B27 expression, and, in some cases CD3 or B7 expression–are acquired.

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“…It amplifies HLA-B*2701-*2706 alleles, which are the most common in the Caucasian population and the most associated with AS. The second PCR used a combination of 3 primers described by Faner et al [42]. It amplifies a larger number of alleles than the first, from HLA-B*2701 to *2724 except for HLA-B*2718 and HLA-B*2723.…”

Section: Methodsmentioning

confidence: 99%

Ultrasonographic Assessment of Enthesitis in HLA-B27 Positive Patients with Rheumatoid Arthritis, a Matched Case-Only Study

et al. 2013

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IntroductionHLA-B27 has a modifier effect on the phenotype of multiple diseases, both associated and non-associated with it. Among these effects, an increased frequency of clinical enthesitis in patients with Rheumatoid Arthritis (RA) has been reported but never explored again. We aimed to replicate this study with a sensitive and quantitative assessment of enthesitis by using standardized ultrasonography (US).MethodsThe Madrid Sonography Enthesitis Index (MASEI) was applied to the US assessment of 41 HLA-B27 positive and 41 matched HLA-B27 negative patients with longstanding RA. Clinical characteristics including explorations aimed to evaluate spondyloarthrtitis and laboratory tests were also done.ResultsA significant degree of abnormalities in the entheses of the patients with RA were found, but the MASEI values, and each of its components including the Doppler signal, were similar in HLA-B27 positive and negative patients. An increase of the MASEI scores with age was identified. Differences in two clinical features were found: a lower prevalence of rheumatoid factor and a more common story of low back pain in the HLA-B27 positive patients than in the negative. The latter was accompanied by radiographic sacroiliitis in two HLA-B27 positive patients. No other differences were detected.ConclusionWe have found that HLA-B27 positive patients with RA do not have more enthesitis as assessed with US than the patients lacking this HLA allele. However, HLA-B27 could be shaping the RA phenotype towards RF seronegativity and axial involvement.

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HLA-B27 genotyping by Fluorescent Resonance Emission Transfer (FRET) probes in real-time PCR

Cited by 20 publications

References 25 publications

HLA‐DQ2/DQ8 and HLA‐DQB102* homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

HLA‐DQ2/DQ8 and HLA‐DQB102* homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

Flow Cytometric Screening for the HLA‐B27 Antigen on Peripheral Blood Lymphocytes

Ultrasonographic Assessment of Enthesitis in HLA-B27 Positive Patients with Rheumatoid Arthritis, a Matched Case-Only Study

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HLA-B27 genotyping by Fluorescent Resonance Emission Transfer (FRET) probes in real-time PCR

Cited by 20 publications

References 25 publications

HLA‐DQ2/DQ8 and HLA‐DQB1*02 homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

HLA‐DQ2/DQ8 and HLA‐DQB1*02 homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

Flow Cytometric Screening for the HLA‐B27 Antigen on Peripheral Blood Lymphocytes

Ultrasonographic Assessment of Enthesitis in HLA-B27 Positive Patients with Rheumatoid Arthritis, a Matched Case-Only Study

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Product

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HLA‐DQ2/DQ8 and HLA‐DQB102* homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population

HLA‐DQ2/DQ8 and HLA‐DQB102* homozygosity typing by real‐time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population