Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

@#Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorders which leads to erythrocytosis, thrombocytosis and leucocytosis. MPN with BCR-ABL positive is chronic myeloid leukaemia (CML) while BCR-ABL negative MPN includes polycythaemia Vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, formalin-fixed paraffin-embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. Materials and Methods: This study is a descriptive crosssectional study. Forty FFPE marrow specimens were retrieved from the years 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. Results: JAK2-V617F mutation was positive in 13 out of 40 MPN cases. Level of agreement between HRM and AS-PCR was 97.5%. Conclusion: HRM is a rapid and powerful diagnostic assay which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

Study on the evolution of the minor resistant mutations and the primary resistance in rural areas of Henan / 中华微生物学和免疫学杂志

Objective To evaluate the antiretroviral therapy(ART),analyze the prevalence of resistance in rural areas,Henan,and explore the presence of minor resistant variants in pre-ART.Methods One hundred and forty-nine AIDS patients initiating ART were recruited and investigated at intervals of 6 months. Method of In-house developed by our laboratory for genotypjc resistance test was to analyze the occurrence of resistance among the failure of ART,and the allele-specific real.time PCR(ASPCR)was used to detect the minor resistant variants at the baseline samples once the resistance occurred.Results Vimlload significantly decreased among the patients who received ART(t=275,P=0.0001),but the absolute counts of CD4+T lymphocytes had no significant change(t=1.765 168,P=0.0852).Rate of resistance among the patients of treatment failure was 4.88%.The result of ASPCR in the survey of baseline showed that the minor resistant variants of M184V were detected in 7 patients and mutation K103N presented in 5 patients.Conclusion The primary drug-resistant straias in the untreated patients were found in Henan,and they might develop the dominant resistance strains and bring about the failure of ART.

Allele-specific real-time PCR for the detection of minor HIV-1 variants / 中华微生物学和免疫学杂志

Objective To develop and evaluate the allele-specific real-time PCR(ASPCR) assay for the detection of minor HIV-1 variants. Methods We developed and evaluated the ASPCR assay, using the K103N mutation site as a model system. We constructed plasmids as standards and designed specific and non-specific primers to discriminate the wild-type and mutant plasmids in the real-time PCR using SYBR green as fluorescence reporter. And then we evaluated the sensitivity, accuracy, reproducibility of ASPCR assay and detected the control samples. Results The specific primer can discriminate the wild-type and mutant plasmids including resistant mutation successfully. The sensitivity of ASPCR assay can achieve less than 0.01% and the accuracy of this method is down to 0.1%. The Intra-assay coefficient of variation is less than 0.7 and the Inter-assay coefficient of variation is less than 1.6. Conclusion ASPCR is a sensitive, accurate and rapid method to detect the minor HIV-1 variants which have resistant mutations and it can be used widely in HIV research. ASPCR also can provide earlier and more resistant information to the clinical therapy.

A Simplified ABO Genotyping by Allele-Specific Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: Genotyping of ABO gene could be more informative and valuable than serological typing in some situations such as the resolution for ABO discrepancy between the cell typing and serum typing and determination of A and B subgroups. We developed a simple allele-specific polymerase chain reaction (AS-PCR) method without the use of any restriction enzymes to detect the A, B, O, and cis-AB alleles for Koreans. METHODS: An AS-PCR was designed with amplification refractory mutation system (ARMS) at nt (nucleotide) 261 (exon 6) and at nt 526, 803 (exon 7) of ABO gene to detect specific nucleotide sequence differences between the ABO alleles. We tested for ABO genotyping 60 DNA samples previously tested by PCR-RFLP and stored at -70degreeC. These samples had been obtained from blood donors recruited at the Gwangju-Chonnam Red Cross Blood Center between July 2002 and February 2003. RESULTS: With our new PCR method, the genotypes of the 60 samples were found to be A/O (n=10), A/A (n=5), B/O (n=10), B/B (n=5), O/O (n=10), cis-AB/A (n=5), cis-AB/B (n=5), and cis-AB/ O (n=10), which were the s ame results obtained previously with PCR-RFLP. CONCLUSIONS: Our AS-PCR is a simple and accurate method for the detection of A, B, O, and cis-AB alleles for Koreans.