Application of Real-Time PCR and Melting Curve Analysis in Rapid Detection of A el and B el Blood Types

Transcription

1 Annals of Clinical & Laboratory Science, vol. 35, no. 1, Application of Real-Time PCR and Melting Curve Analysis in Rapid Detection of A el and B el Blood Types Ding-Ping Chen, 1 Ching-Ping Tseng, 2 Hung-Tse Lin, 2 and Chien-Feng Sun 1 1 Department of Clinical Pathology, Linkou Medical Center, Chang Gung Memorial Hospital, and 2 Graduate Institute of Medical Biotechnology and School of Medical Technology, Chang Gung University, Taoyuan, Taiwan Abstract. The ABO blood group is the most important blood group system in transfusion medicine. In addition to the normal levels of ABO antigen expression, A el and B el represent the two major blood types that have a weak expression of the A or B antigens on red blood cells. Due to the fact that typing of A el and B el by conventional serological methods is time consuming and sometimes gives false-positive and falsenegative results, it is warranted to develop an additional technique for the identification of A el and B el individuals. Through genetic analysis we have previously identified A el as possessing an A allele with IVS6+5G A mutation (Transfusion 2003;43: ) and B el as possessing a B gene with 502C T mutation in Taiwan (Vox Sanguinis 2003;85: ). Hence, real-time PCR-based genotyping methods were developed in this study to facilitate the detection of A el and B el. For genotyping of A el and B el, the region of mutations was PCR amplified and subjected to the LightCycler (LC) real-time PCR assay using LC Red640-labeled hybridization probe. Melting curve analysis was performed to determine the melting temperature T m that was used for genotype detection of A el and B el blood types. For A el genotyping, the melting curve of the normal control appears as one peak at ± 0.07 C (mean ± SE) and that of A el appears as 2 peaks at ± 0.07 C and ± 0.07 C, corresponding to the O and A el alleles, respectively. For B el genotyping, the melting curve of the normal control appears as one peak at ± 0.11 C and that of B el appears as 2 peaks at ± 0.12 C and 68.1 ± 0.13 C, corresponding to the B el and O alleles, respectively. This genotyping method was shown to be accurate, based on automated sequencing of the PCR-amplified products. It takes only 90 min to perform this genotyping test. Detecting the A el and B el blood types by combined LC-PCR and melting-curve analysis is a rapid, reliable, and easy method. (received 29 September 2004; accepted 5 October 2004) Keywords: A el blood group, B el blood group, genotyping, real-time PCR, melting curve analysis Introduction The ABO blood group is the most important blood group system in transfusion medicine. In addition to the common ABO blood types, distinct blood types with a weak expression of the A or B antigens on the red blood cells (RBC) have been identified and were designated as A 3, A x, A el, B 3, B x, B el, cis- Address correspondence to Chien-Feng Sun, M.D., Department of Clinical Pathology, Linkou Medical Center, Chang Gung Memorial Hospital, 5 Fushin Street, Kweishan, Taoyuan, 333 Taiwan, ROC; tel , ext. 2554; fax ; suncgj@adm.cgmh.org.tw. AB, and B(A), respectively [1,2]. Population study has revealed that A el and B el are the 2 major weak A and weak B blood types in Taiwan [3]. These blood types are generally identified by the adsorptionelution test and saliva test. However, these methods are time-consuming and the results are sometimes confusing. For instance, false-positive results may be obtained due to cold agglutinins, bacterial agglutinins, or over-centrifugation. False-negative results may be caused by low-titer antisera or a newborn s immature RBCs. Therefore an alternative approach is warranted for the identification of A el and B el individuals /05/ $ by the Association of Clinical Scientists, Inc.

2 26 Annals of Clinical & Laboratory Science, vol. 35, no. 1, 2005 Mutational alterations of the ABO genes account for distinct A/B blood subtypes. These genetic mutations are inheritable and usually occur in the ABO gene coding sequence or the consensus sequences located on the mrna splicing site. In addition, most of the mutations are single-nucleotide substitutions, leading to an amino acid alteration. During the past few years, we analyzed the genetic changes that are responsible for the weak A and weak B phenotypes. Our data showed that individuals with A el possess an A allele with the IVS6+5G A mutation [4], whereas individuals with B el carry a B allele with the 502C T mutation [5]. Besides, Taiwanese individuals with the B 3 phenotype carry a B allele with a G A mutation at the +5 nucleotide of intron 3 [6]. These data suggest strong association of a specific ABO allelic change with a specific ABO blood type and enable us to develop genotype-based methods for the identification of distinct A and B blood subgroups in Taiwan. Several PCR-based techniques have been developed for genotyping analysis of various clinical specimens. Among these methods, the real-time PCR technique has proven useful in allelic discrimination. This can be achieved by use of an allelespecific TaqMan probe [7], molecular beacon [8], or hybridization probe, followed by analysis of allelespecific melting behavior [9]. Fluorescence monitoring using hybridization probes is based on the principle that a fluorescence signal is generated if fluorescence resonance energy transfer (FRET) occurs between two adjacent fluorophores [10]. This detection process allows monitoring of the intensity of the FRET signal, which is proportional to the amount of specific PCR product generated. More important, genotyping using 2 hybridization probes is possible with a detection probe that spans the polymorphic site and an anchor probe that recognizes an adjacent sequence, since polymorphic alleles can be distinguished by the melting temperature (T m ) of the detection probe. Continuous fluorescence monitoring of the reaction as the temperature is raised from annealing to denaturation results in a sharp decrease in fluorescence at the temperature at which the detection probe dissociates from the template. The single base change caused by ABO polymorphism results in a decrease of T m of the detection probe that can be distinguished readily using the LightCycler instrument. In this study, we describe the development of a hybridization probe-based real-time PCR technique for the detection of A el and B el genotypes. The realtime PCR was performed in the LightCycler thermal cycler and the allele-specific melting behavior of the fluorophore-labeled hybridization probe was used to detect A el - and B el -specific genotypes. The method is evaluated by clinically available specimens. Using this method will facilitate the identification of individuals with the A el or B el blood types and could be integrated into routine typing of rare blood types in clinical diagnostic laboratories. Materials and Methods Specimen collection and genomic DNA isolation. Through serological screening, a total of 7 unrelated individuals with the A el blood type, 8 unrelated individuals with the B el blood type, 10 individuals with the common A 1, and another 10 individuals with the common B blood types were identified at Chang Gung Memorial Hospital. Genomic DNA was prepared from their peripheral blood cells using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). Real-time PCR and melting curve analysis for genotyping of A el and B el. Real-time PCR was set up for the amplification of exons 6 and 7 of the ABO gene. Briefly, 1 ng of genomic DNA was added to the reaction mixtures (20 µl) containing 1x LightCycler FastStart DNA Master Hybridization Probe buffer (dntp, Taq DNA polymerase, and 5 mm Mg 2+ ), 500 nm forward and reverse primers, 250 nm 3 -FL-labeled detection probe, and 250 nm 5 -LC-labeled anchor probe. Reaction mixtures were loaded in glass capillary cuvets (Roche Molecular Biochemicals, Mannheim, Germany) and were centrifuged to place the sample at the capillary tip before capping. After an initial denaturation step at 95 C for 10 min, amplification was performed by using 50 cycles of denaturation (95 C for 10 sec), annealing (55 C for 10 sec), and extension (72 C for 20 sec) on a LightCycler fluorometric thermal cycler (Roche Molecular Biochemicals). The

3 Rapid detection of A el and B el blood types 27 Fig 1. For A el typing, the sense primer (blue color; nt , locates at exon 6) and the antisense primer (green color; nt , locates at intron 6) were used to amplify a 228 bp fragment of the A el gene, harboring the polymorphic site at nucleotide 4103 (IVS6+5G A; as arrow indicates). The detection probe was a 25-mer oligonucleotide (yellow color; nt ) labeled at the 3 -end with Light- Cycler Red 640. The 5 -fluorescein labeled anchor probe (red color; nt ) was a 23- mer that binds with a distance of one base to the detection probe. The sequence of the detection probe was chosen in such a way that it was not complementary to another normal A allele. temperature transition rates were 20 C/sec from denaturation to annealing, annealing to extension, and extension to denaturation. Fluorescence was measured at the end of the annealing period of each cycle to monitor the concentration of amplicon. After amplification was complete, a final melting curve was recorded by heating to 95 C for <1 sec and then cooling to 50 C at 20 C/sec, followed by a 60-sec hold before heating slowly at 0.1 C/sec until a temperature of 95 C was attained. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled detection probe. The fluorescence signal (F) was plotted in real-time against the temperature (T) to produce melting curves for each sample (F versus T). Melting curves were converted to melting peaks by plotting the negative derivative of F with respect to T against T (-df/dt vs T). The entire process required about 30 min. Genotyping by direct sequencing. For confirmation of genotypes, the DNA fragment encompassing exon 6 through exon 7 of the ABO gene was PCR amplified and sequenced directly as described previously [6,7]. Briefly, 100 ng of genomic DNA was added to reaction mixtures containing 25 µl of PCR buffer (0.2 mm of dntp and 0.5 unit of Expand HiFi PLUS DNA polymerase, Roche Diagnostics), 10 pmole of forward primer: (5 -GGGTGGTCAGAGGAGGCAGAAGCTGAGTGG-3 ) that located at 91 bp upstream to exon 6, and 10 pmole of reverse primer: (5 -GACGGGGCCTAGGCTTCAGTTACTCACAAC -3 ) that located at 99 bp downstream to the stop codon. The PCR program consisted of 5 min at 94 C followed by 30 cycles of 0.5 min at 94 C and 2.5 min at 72 C. The 2.1 kb PCR product was used as the templates for DNA sequencing using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Results A el possesses an A allele with an IVS6+5G A mutation at the intron 6, whereas B el carries a B allele with the 502C T mutation at the exon 7. To facilitate the identification of individuals with the A el and B el blood types, a genotyping technique was developed using the LightCycler real-time PCR and melting curve analysis. Primers and hybridization probes were designed for real-time PCR

4 28 Annals of Clinical & Laboratory Science, vol. 35, no. 1, 2005 Fig. 2. For Bel typing, the sense primer (blue color; nt , locates at exon 7) and the antisense primer (green color; nt , locates at exon 7) were used to amplify a 154 bp fragment of the Bel gene, harboring the polymorphic site at nucleotide (502C T). The detection probe was a 20-mer oligonucleotide (yellow color; nt ) labeled at the 3 end with LightCycler Red 640. The 5 -fluorescein labeled anchor probe (red color; nt ) was a 23-mer that binds with a distance of one base to the detection probe. The sequence of the detection probe was chosen in such a way that it was not all complementary to Bel allele. amplification of intron 6 of the A allele and exon 7 of the B allele, respectively (Figs. 1,2). As expected, the PCR products with 228 bp for A allele and 154 bp for B allele were obtained after amplification of the genomic DNA from individuals with A and B blood types (data not shown). Melting curve analysis of the A allele PCR product revealed that individuals with normal A blood type had a single melting peak with Tm = ± 0.07 C (mean ± SE). The individuals with Ael blood type had 2 melting peaks with Tel = ± 0.07 C and ± 0.07 C, and corresponded to the O and Ael allele, respectively (Fig. 3). Analysis of the B allele PCR product revealed that individuals with normal B blood type had a melting peak with Tm = ± 0.11 C. In contrast, individuals with Bel had 2 melting peaks with Tm = ± 0.12 C and 68.1 ± 0.13 C, and corresponded to the Bel and O alleles, respectively. All the 7 Ael, 8 Bel, 10 A1, and 10 B individuals were correctly identified by the real-time PCR and melting curve analysis. In addition, the accuracy of this genotyping method was confirmed by automated sequencing of the PCR amplified products. Discussion The Ael and Bel blood types account for 80.03% and 100% of the weak A and weak B Taiwanese individuals, respectively. The identification of Ael and Bel phenotypes has traditionally relied on serological phenotyping methods, including the adsorption and elution test. These techniques are time-consuming and are crucially dependent on the quality of testing to obtain validated data. Determination of Ael and Bel phenotypes at the nucleic acid level represents an alternative approach. Several PCR-based methods, including multiplex SSP [11], RFLP [5,12,13], and SSCP [14-17] have been reported for genotyping of the ABO blood groups. In the present study, we report a novel method for blood typing of potential Ael and Bel individuals. This technique is based on the use of hybridization probe-based real-time PCR technique and melting curve analysis to determine whether the Ael or Bel genotype is present in the ABO alleles of the test subject. This method has several advantages in comparison with the serological methods. The real-

5 Rapid detection of A el and B el blood types 29 Fig. 4. Work-flow chart that can replace the absorption-elution test, which is time-consuming and less accurate. Fig 3. Melting curve analysis to detect A el and B el blood types. time PCR genotyping technique is more precisely and clearly revealed by the analysis of melting curve profile. In contrast, the serological blood typing method usually depends on the objective observation of RBC agglutination and may result in misreading of the blood type. The real-time PCR has better sensitivity and specificity; usually <1 ml of peripheral blood is required for the test. Since blood typing of A el and B el is achieved within 90 min after DNA extraction, this also provides a rapid method for blood typing of A el and B el. It is noted that the genotypes of A el and B el are different in various regions and countries. For instance, two different molecular changes, 804insG and 646T A in the A allele, have previously been reported to be responsible for the A el phenotype [18,19]. Mutations of 641T G and 669G T, respectively, in the B allele have been reported for the B phenotype in other ethnic groups [20]. Therefore, it is necessary for others to formulate a similar genotyping method according to the regional mutation occurrence of the A el and B el blood types. Our analyses indicate that all A el individuals in Taiwan have an A allele with IVS6+5G A mutation, whereas all the B el individuals have a B gene with the 502C T mutation. Hence, we propose a new work-flow chart for rare blood typing (Fig. 4). First, a serological method is used for rapid screening for the presence of ABO antigen and antibody. When the result of forward blood type O and reverse blood type A or B is obtained, the genomic DNA of the peripheral blood is extracted for real-time PCR and melting curve analysis to determine whether the test subject has the A el or B el blood type. In conclusion, real-time A el and B el genotyping using LightCycler technology combined with

6 30 Annals of Clinical & Laboratory Science, vol. 35, no. 1, 2005 melting-curve analysis is a fast, simple, cost-effective and convenient method for rare blood typing of A el and B el. This technique could readily be integrated into routine blood typing in a clinical diagnostic laboratory. Acknowledgement Technical assistance by the Tib Molbiol Co., Berlin, Germany, is gratefully acknowledged. References 1. Issitt PD, Anstee DJ. Applied Blood Group Serology. Montgomery Scientific, Durham, NC, Chester MA, Olsson ML. The ABO blood group gene: a locus of considerable genetic diversity. Transfusion Med Rev 2001;15: Lin M, Broadberry RE. Immunohematology in Taiwan. Transfusion Med Rev 1998;12: Sun CF, Yu LC, Chen DP, Chou ML, Twu YC, Wang WT, Lin M. Molecular genetic analysis for the A el and A 3 alleles. Transfusion 2003;43: Sun CF, Chen DP, Lin KT, Wang WT, Wang YC, Yu LC. Molecular genetic analysis of the B el phenotype. Vox Sang 2003;85: Yu LC, Twu YC, Chou ML, Chang CY, Wu CY, Lin M. Molecular genetic analysis for the B 3 allele. Blood 2002; 100: Ariani F, Mari F, Pescucci C, Longo I, Bruttini M, Meloni I, Hayek G, Rocchi R, Zappella M, Renieri A. Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome. Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat 2004;24: Root DD, Vaccaro C, Zhang Z, Castro M. Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer. Biopolymers 2004;75: Pont-Kingdon G, Lyon E. Rapid detection of aneuploidy (trisomy 21) by allele quantification combined with melting curves analysis of single-nucleotide polymorphism loci. Clin Chem 2003;49: Morrison LE. Detection of energy transfer and fluorescence quenching. In: Nonisotopic DNA Probe Techniques (LJ Kricka, Ed), Academic Press, San Diego, CA, Downing J, Darke C. A modified PCR-SSP method for the identification of ABO blood group antigens. Eur J Immunogenet 2003;30: Ishida K, Zhu B, Sakoda S, Quan L, Oritani S, Fujita MQ, Maeda H. Significance of DNA analysis for determination of ABO blood groups from hair and nail of decomposed human remains: a comparison with phenotyping by the absorption-elution method. Legal Med (Tokyo) 2000;2: Hummel S, Schmidt D, Kahle M, Herrmann B. ABO blood group genotyping of ancient DNA by PCR-RFLP. Int J Legal Med 2002;116: Olsson ML, Chester MA. Polymorphism and recombination events at the ABO locus: a major challenge for genomic ABO blood grouping strategies. Transfusion Med 2001;11: Tsai LC, Kao LG, Chang JG, Lee HH, Linacre A, Lee JC. Rapid identification of the ABO genotypes by their single-strand conformation polymorphism. Electrophoresis 2000;21: Yip SP. Single-tube multiplex PCR-SSCP analysis distinguishes 7 common ABO alleles and readily identifies new alleles. Blood 2000;95: Diamond DC, Illes K, Bailey LL, Szalay AA. Genotyping the baboon ABO histo-blood group locus by two-color fluorescence SSCP. Biotechniques 1999;27: , , Olsson ML, Thuresson B, Chester MA. An A el allelespecific nucleotide insertion at the blood group ABO locus and its detetion using a sequence-specific polymerase chain reaction. Biochem Biophys Res Commun 1995; 216: Ogasawara K, Yabe R, Uchikawa M, Nakata K, Watanabe J, Takahashi Y, Tokunaga K. Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes. Immunogenetics 2001;53: Ogasawara K, Yabe R, Uchikawa M, Saitou N, Bannai M, Nakata K, Takenaka M, Fujisawa K, Ishikawa Y, Juji T, Tokunaga K. Molecular genetic analysis of variant phenotypes of the ABO blood group system. Blood 1996;88: