Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.
DNA Primers ; Formaldehyde ; Gene Expression Profiling ; Humans ; MicroRNAs/analysis* ; Myocardium ; Paraffin Embedding ; RNA/analysis* ; Real-Time Polymerase Chain Reaction/standards*

OBJECTIVE: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). METHODS: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. RESULTS: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. CONCLUSION The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.
Anion Exchange Protein 1, Erythrocyte ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Heterozygote ; Humans ; Mutation ; Spherocytosis, Hereditary ; genetics

OBJECTIVETo provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

METHODSViruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

RESULTSNGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

CONCLUSIONThe improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

OBJECTIVETo analyze a family where some members were suspected as rare ABw31 subtype.

METHODSSerological assay was performed to identify the ABO blood group of the proband and other family members. Genotypes for exons 6 and 7 of the ABO gene were determined with sequence-specific primer polymerase chain reaction (SSP-PCR) and direct sequencing.

RESULTSBoth A and B antigens were detected on the red blood cells from the proband. There was also anti-A antibody in the serum. The serological phenotype of the sample was identified as ABw. DNA sequencing confirmed heterozygous status of 297AG, 467CT, 526CG, 657CT, 664GA, 703AG, 796AC, 803GC, and 930GA for exons 6 and 7. Clone of two alleles (A102 and Bw31) were obtained. Compared with the B101 allele, sequence of Bw31 allele showed one nucleotide change (G to A) at position 664, which resulted in replacement of Val with Met at position 222. The proband's father, brother and son all carried the Bw31 allele.

CONCLUSIONThe 664G>A mutation probably underlies the Bw phenotype and can be transmitted stably.

ABO Blood-Group System ; genetics ; Adult ; Base Sequence ; DNA Primers ; Exons ; genetics ; Family Health ; Female ; Genotype ; Humans ; Male ; Mutation, Missense ; Pedigree ; Phenotype ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods

OBJECTIVETo explore the molecular basis for a CD36 deficiency individual and distribution of CD36 gene mutation in Guangxi population.

METHODSA female individual was studied. CD36 phenotype was detected by monoclonal antibody immobilization of platelet antigens assay (MAIPA) and flow cytometry (FCM). The coding regions of the CD36 gene were sequenced. A DNA-based polymerase chain reaction-sequence specific primer (PCR-SSP) assay was used to verify the identified mutation. Cell lines expressing the mutant and wild-type CD36[CD36(MT) and CD36(WT)] were established, with the expression of CD36 determined by Western blotting. The distribution of CD36 gene mutation was investigated among 1010 unrelated individuals with the PCR-SSP assay.

RESULTSBoth MAIPA and FCM assays showed that the patient had type II CD36 deficiency. DNA sequencing showed that she has carried a heterozygous mutation T538C (Trp180Arg) in the exon 6 of CD36. Sequencing of cDNA clone confirmed that there was a nucleotide substitution at position 538 (538T>C). Western blotting also confirmed that the CD36 did not express on the CD36(MT) cell line that expressed the 538C mutant, but did express on the CD36(WT) cell line. The novel CD36 mutation T538C was further verified with 100% concordance of genotyping results by DNA-based PCR-SSP assay and 1010 unrelated individuals. No CD36 538C allele was detected among the 1010 individuals.

CONCLUSIONThis study has identified a novel CD36 mutation T538C(Trp180Arg)(GenBank: HM217022.1), and established a genotyping method for the novel sequence-specific primer PCR. The novel mutation is rare in Guangxi and can cause type II CD36 deficiency.

Base Sequence ; Blood Platelet Disorders ; genetics ; Blood Platelets ; cytology ; metabolism ; Blotting, Western ; CD36 Antigens ; genetics ; metabolism ; Cells, Cultured ; DNA Mutational Analysis ; DNA Primers ; genetics ; Exons ; genetics ; Female ; Flow Cytometry ; Fluorescent Antibody Technique ; Genetic Diseases, Inborn ; genetics ; Genotype ; Genotyping Techniques ; methods ; Humans ; Middle Aged ; Monocytes ; cytology ; metabolism ; Mutation, Missense ; Polymerase Chain Reaction ; methods

OBJECTIVE: To explore the change rules of peak area ratio of STR loci to Amelogenin (AMEL) locus (STR/AMEL), a sex-determining gene in DNA degradation, and to evaluate the application of STR/AMEL value in the estimation of DNA degradation degree. METHODS: DNA was extracted from iliopsoas, and the variations of STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) were analyzed after the artificial degradation was made by DNase I, and the changes of these three ratios of the iliopsoas naturally degraded in an outdoor environment were also analyzed. The regression curves were analyzed using the periods of DNA degradation and outside the body as the independent variable (x) and the STR/AMEL value as the dependent variable (y) and three curve equations under two conditions were established. RESULTS: Both under the conditions of artificial and natural degradation, STR/AMEL value had a negative relationship with the degradation time. The relationship between STR/AMEL and degradation time can be well simulated by the cubic function. R2 was over 0.99 under controlled degradation condition and over 0.86 under natural degradation condition. CONCLUSION The STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) is negatively related with the DNA degradation degree, which follows mathematical regression models strictly, and it might be applied to evaluate the DNA degradation degree.
Amelogenin/genetics* ; DNA Damage/genetics* ; DNA Primers ; Humans ; Microsatellite Repeats ; Regression Analysis ; Time Factors

BACKGROUND: Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method. METHODS: We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line. RESULTS: The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%). CONCLUSIONS: The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.
Adult ; Aged ; Asian Continental Ancestry Group/genetics ; Biopsy, Fine-Needle ; DNA/chemistry/metabolism ; DNA Mutational Analysis/*methods ; DNA Primers/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Multiplex Polymerase Chain Reaction ; Oligonucleotides/metabolism ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins B-raf/*genetics ; Republic of Korea ; Thyroid Nodule/*metabolism/pathology

OBJECTIVETo establish a method for detecting 3 common toxigenic molds (Aspergillus, Penicillium, and Fusarium) based on non-modified magnetic beads coupled with multiple real-time PCR (NMB-multiple qPCR).

METHODSThe primers and genus-specific probes were designed based on the rDNA sequences to develop a multiple real-time PCR using non-modified magnetic bead to enrichment of fungal spores. The sensitivity, specificity and repeatability of this assay were evaluated.

RESULTSThe detection limit of this assay for spiked samples was 10(4) CFU/g, demonstrating a 10-fold greater detection sensitivity of this assay than that of real-time PCR. The NMB-multiple qPCR assay also showed good specificity and reproducibility and yielded comparable results with those by traditional colony counting method for spiked samples (P>0.05).

CONCLUSIONNMB-multiple qPCR assay we established allows rapid and sensitive detection of common mycotoxigenic fungi in paprika.

Aspergillus ; Capsicum ; microbiology ; DNA Primers ; Food Contamination ; analysis ; Food Microbiology ; Fungi ; isolation & purification ; Fusarium ; Magnetic Phenomena ; Penicillium ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo identify a novel HLA allele DRB1 * 16:36 from a Uygur woman.

METHODSPCR-SBT technology was applied to the extracted DNA for genotyping, and a possible new gene was sequenced by using sequence specific primers and single stranded SBT. This novel allele was compared with known most homologous gene sequences and their difference was analyzed.

RESULTSThis novel allele was different from HLA alle DRB1 * 16:23, and had highest similarity in 2 nucleotides at position 227 A→T and 236 T→C in exon 2, resulting in 3 amino acid changes from Tyr to Phe at codon 47 and Val to Ala at codon 50. The sequence of this novel allele had been submitted to GenBank.

CONCLUSIONThis HLA allele DRB1 * 16:23 has been confirmed to be a novel allele, and has been officially named DRB1 * 16:36 by the World Health Organization (WHO) Nomenclature Committee in May 2015.

Alleles ; Base Sequence ; DNA Primers ; Exons ; Female ; Genotype ; HLA-DRB1 Chains ; genetics ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo explore the molecular mechanism for a case with para-Bombay phenotype caused by α-1,2-fucosyltransferase (FUT1) gene mutations.

METHODSBlood phenotype of the propositus was determined by standard serological testing. Polymerase chain reaction-sequence specific primer (PCR-SSP) and direct sequencing of PCR product were used to analyze its ABO genotype. The PCR product of FUT1 gene was sequenced and analyzed.

RESULTSThe phenotype of the propositus was initially detected as para-Bombay A type. Direct sequencing of ABO gene showed that the genotype of the proband was A101/O01 (261G/del), which was consistent with the result of PCR-SSP. Two homo-mutations, 35C>T and 658C>T, were detected in the FUT1 gene by sequencing, and the genotype was determined as h(35T+658T)/h(35T+658T).

CONCLUSIONh(35T+658T)/h(35T+658T) is responsible for the para-Bombay phenotype of the propositus. The genotype is rare even in para-Bombay populations.

ABO Blood-Group System ; genetics ; Base Sequence ; DNA Mutational Analysis ; methods ; DNA Primers ; Fucosyltransferases ; genetics ; Genotype ; Homozygote ; Humans ; Male ; Phenotype ; Point Mutation ; Polymerase Chain Reaction