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Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples,and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection.Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database,and fixed specific oligonucleotide probes on the nylon membrane.After multiple PCR amplification of virus DNA of human bocavirus (hBOV),karolinska Institutet (KI),adenovirus (AdV),Washington University polyomaviros (WUPyV),and human parvovirus B19 (HPVBI9),the denaturalized amplification products were hybridized with various specific probes,followed by visualization and analysis of the results.The sensitivity and specificity were tested.At the same time,108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection,and compared to the results of culture method.Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen.The sensitivity of RDB hybridization was 1 colony-forming units (CFU).The positive rate of 34.26％ (37 cases out of 108 cases) with PCR-RDB method was significandy higher than that 27.78％ (30 cases out of 108 cases) with common test method.Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples,which might be a promising tool for clinical application.
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BACKGROUND:In recent years, with the development of China Marrow Donor Program and the improvement of human leukocyte antigen (HLA) typing technique, novel aleles of human leukocyte antigen have been discovered constantly in China. OBJECTIVE:To identify and confirm a novel HLA alele in a Chinese individual. METHODS: A new HLA alele was found during routine human leukocyte antigen genotyping by PCR-sequence specific oligonucleotide probes and sequencing-based typing. RESULTS AND CONCLUSION:The HLA-B locus hybridization probe reaction patterns of this sample did not match with any known HLA-B aleles or alelic combinations. Exons 2, 3 were sequenced in both directions using HLA-B sequence primer and group-specific sequencing primer. The obtained sequence had 2nts change from B*07:02:01 at nt 226 and nt 228 where A->G (codon 76 ATA->GTG) and resulting in a coding change, 76 isoleucine (I) was changed to valine (V). This nucleotide sequence has been submitted to the GenBank nucleotide sequence database and it is available under the accession number HM989017. A novel HLA alele, HLA-B*07:110, was identified, and was named officialy by the WHO Nomenclature Committee.
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Objective Establishment and development of a novel Single-Nucleotide-Polymorphism TaqMan Real-Time PCR assay for rapid detection of rs12979860 that predicts HCV therapy response.Methods Human genomic DNA were extracted from solid tissues,secretion and plasma before allelic discrimination.With the property of minor groove binding protein (MGB) binding to minor groove of DNA with strong specificity and affinity,primers and MGB probes were particularly designed for differentiation of human genomic frequencies.MGB probe-based real-time PCR was established to increase allelic discrimination using two probes that only differ in one nucleotide of IL28B rs12979860.The specificity was evaluated by fluorescence signal emissions which were selected from two signal channels.And DNA sequencing was used to confirm the genomic polymorphisms.Results TaqMan probe-based SNP real-time PCR increased allelic discrimination using two probes that only differ by one nucleotide of amplicon,which indicated this assay was easily performed regardless of genomic DNA concentration and quality,minimizes sources of error.The sensitivity was as low as 1.5 ng/μl,the amplification efficacy was 97.6％.The genotype frequencies of CC,CT were remarkably different between Caucasian and Mongolian.The dominated genotype of Caucasian was CT,while most Mongolian was carried CC (26/40 vs.40/50,x-2 =18.75,P value ＜ 0.05).However,the genotype between two population showed no relationship with their virological clearance clinically (P value ＞ 0.05).Conclusions This TaqMan MGB assay shows highly specificity,which has potential as a routine diagnostic test for the detection of rs12979860 from various types of samples.This robust assay would be widely used clinically to predict patients' response before anti-HCV treatment in personalized medicine.
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Objective To investigate the genotype distribution of hepatitis C virus (HCV) in Guangdong Province by linear probe assay (LiPA) and core,non-structural protein 5B (NS5B) sequence analysis,and to evaluate the accuracy of LiPA for genotyping.MethodsOne hundred and ten HCV specimens from chronic hepatitis C (CHC) patients in Guangdong Province were genotyped by both core,NS5B sequence analysis and INNO-LiPA 2.0.The data were then analyzed with SPSS 10.0 software.ResultsAmong the 110 specimens,core and NS5B fragment could be amplified from 97 and 62 specimens,respectively,while both fragments were obtained from 57 specimens. The results of genotyping by core sequence analysis were completely consistent with the results of NS5B sequencing.One hundred and two specimens were classified into five subtypes,i.e.genotype 1a,2a,3a,3b,6a,with frequencies of 61.8％ (64),9.8％ (10),3.9％ (4),3.9％ (4),20.6％ (21),respectively.All but genotype 6 strains can be genotyped correctly by using INNO-LiPA 2.0.However,only subtype 1b and 3b could be genotyped accurately.81.5％ genotype 6a strains were genotyped as 1b by mistake. Conclusions Genotype 6a has become the second most prevalent genotype in Guangdong Province.The LiPA cannot distinguish genotype 6a from 1b strains accurately which needs further investigation.
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Objective To investigate the effect of different amplification methods and probes with various length on the results of comparative genomic hybridization (CGH) analysis of pre-implanted single blastomere and to establish the basis for preimplantation genetic diagnosis.Methods Twenty blastomeres of embryo at 6-8 cells stage were randomly divided into A and B group with 10 in each.Twenty peripheral blood lymphocytes from a healthy man were similarly divided into C and D group with 10 in each.Degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) was used to amplify whole genomic DNA in group A and C,and multiple displacement amplification (MDA) was used in group B and D for whole genome amplification (WGA).The specificity of resultant products was confirmed by amplification of TBX1 gene exon 2.CGH was performed respectively with 250-750 bp and 750-2000 bp probes prepared from the amplified whole genomic DNA.The result of CGH was verified by sex-determining region of Y (SRY).Results (1) Nine of the 10 samples in group A and all in group C were amplifiable by DOP-PCR,but there were multiple non-specific bands in the amplification of TBX1 exon 2 when WGA products were used as templates.When 250～750 bp probe was used in CGH,1 of the 5 blastomeres was failed and another one had different karyotype from that analyzed by SRY.(2) All samples in group B and D were successfully amplified by MDA,and the non-specific bands were significantly less in the amplification of TBX1 exon 2.All 5 blastomeres were successful in CGH with the 250～750 bp probe.Moreover,the karyotype was in agreement with that of SRY.(3) When 750 ～ 2000 bp probe was used,the CGH results were suboptimal.Conclusions In WGA of single blastomere,MDA is superior to DOP-PCR in the stability and specificity.The karyotype image detected by CGH with the 250～750 bp probe is clear and homogenous.
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ncing analysis validated that all four-type base-quenched probes could provide unbiased genotyping results ( Kappa =1, P=0.00), although. Conclusion This method is simple, economic and suitable for large-scale genotyping studies.
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Objective To explore the efficacy of multiplex ligation-dependent probe amplification (MLPA)combined with fluorescence in situ hybridization(FISH)and comparative genomie hybridization (CGH)combined with FISH in genetic analysis of chorionic villi specimen(CVS)of spontaneous abortion.Methods CGH+FISH and MLPA+FISH were used for genetic analysis of 29 CVS from spontaneous abortion and 6 normal CVS from selective abortion,in the mean time,those results were compared with conventional eytogenetic karyotyping.Results The report time were 40 hours in MLPA+FISH and 120 houm in CGH+FISH.The mean time of chorionic villi culture was(240±72)hours.The successful rate of specimen analysis were 97%(34/35)in CGH,100%(35/35)in MLPA,100%(35/35)in FISH and 91%(32/35)in conventional cytogenetic karyotyping.Apart from 1 case failed in CGH analysis,the results from MLPA+FISH were almost similar to that from CGH+FISH,however,that 1 specimen failed in CGH were detected successfully by MLPA+FISH.The discrepancy rate were 13%(4/31)in CGH+FISH and 12%(4/32)in MLPA+FISH respectively when compared with conventional cytogenetic analysis.Conclusions MLPA+FISH analysis present shorter detecting time and achieve 100%tale of successful report.This combined method was an important adjuvant approach to conventional cytogenetic karyotyping in CVS from spontaneous abortion.
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Objective To evaluate the clinical value of multiplex ligation-dependent probe amplification (MLPA) technique used in karyotype analysis of chorionic villi from missed abortion. Methods Feb 2008 to Oct 2008, 91 patients with missed abortion diagnosed by hormonal measurement, type B ultrasound and physical exam matched with 20 normal pregnant women undergoing artificial abortion were enrolled in this study. Chorionic villi was obtained by suction dilation and curettage in aseptic condition, then those villi was cultured and analyzed by traditional cytogenetic karyotyping method, in the mean time, the DNA extracted from villi was detected by MLPA. The results of chromosomal G-banding of chorionic villi were compared between two methods. Results The diagnostic concordance of MLPA and traditional karyotyping was observed in 92% (84/91) cases, there were 84 cases in the case group with diagnostic concordance by traditional karyotyping and MLPA except 7 cases of euploidy could not be detected by MLPA. The 84 cases included 40 normal karyotype,29 trisomy of euchromosome, 1 double trisomy of euchromosome, 10 monosomy X , 1 monosomy X combined with trisomy of euchromosome, 2 chimaera of X chromosome, 1 structural abnormity of euchromosome. Among 7 cases with discordance diagnosis, 2 cases with trisomy and 5 cases with tetrasomy of euchromosome were identified in traditional karyotyping, however, they were all diagnosed with normal disomy by MLPA. Of 20 villi from normal pregnancy, two methods got the consistent results. Conclusion The MLPA was rapid and efficacy method used for analyzing aneuploids in chorionic villi.
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Objective To analyze and determine the clinical, molecular pathology and genetic features of a Chinese family with dystrophinopathy. Methods Clinical data of the proband and his family members were collected. Immunohistochemistry staining was performed on muscular biopsy tissues with antimerosin, emerin and the N, C and central rod domains of dystrophin. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes. Multiplex ligation-dependent probe amplification (MLPA) was used to test Duchenne muscular dystrophy (DMD) gene to determine the ways and sites of genetic mutation, and analyze the relationships between genotype and phenotype. Results Patients from this family were clinically diagnosed as muscular dystrophy, and they presented serious manifestations although the immunohistochemistry analysis for the proband exhibited partial loss of dystrophin staining, and positive expression with merosin and emerin. Further test with MLPA detected the loss of exons 45--54 in DMD gene in the proband, while his mother had heterozygositic loss in exons 45--54. Conclusions The losses of exons 45--54 in the proband are all derived from his mother, who carries genetic mutation with normal phenotype. He has been diagnosed as dystrophinopathy. At the same time, his partial loss of dystrophin is not parallel to the out-of-frame mutation of the gene and his severe clinical manifestations. Abnormal expression of dystrophin is the pathological basis for dystrophinopathy phenotype. Its clinical outcome depends not only on the degree of the protein expression, but also on the function of the sites where the DMD gene less occurs.