Diagnóstico molecular de tuberculosis multidrogorresistente en muestras de esputo mediante el análisis de curvas de melting / Molecular diagnosis of multidrug-resistant tuberculosis in sputum samples by melting curve analysis

RESUMEN Objetivos. Analizar curvas de melting para el diagnóstico de tuberculosis multidrogorresistente a partir de muestras de esputo. Materiales y métodos. Se colectaron muestras de esputo (n = 250) de pacientes con sospecha clínica de tuberculosis pulmonar según resultado de baciloscopia y cultivados en medio sólido Lowenstein Jensen. Según el método de referencia se trabajó con 124 muestras sensibles a rifampicina e isoniacida, 24 resistentes a rifampicina, 33 resistentes a isoniacida y 69 multidrogorresistentes. Se evaluó por PCR en tiempo real y luego por las curvas de melting, se utilizó el gen rpoB como biomarcador de resistencia a rifampicina, y el gen katG y región promotora inhA como biomarcadores de resistencia a isoniacida. La cepa H37Rv fue considerada como control sensible a drogas. Se compararon los resultados del método de referencia y los resultados del análisis de curvas de melting para evaluar los parámetros de sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo. Resultados. La resistencia a rifampicina mostró una sensibilidad de 90,3 %, especificidad de 90,4 %, valor predictivo positivo de 84,8 % y valor predictivo negativo de 94,0 %. La resistencia a isoniacida mostró una sensibilidad de 90,2 %, especificidad de 93,9 %, valor predictivo positivo de 91,1 % y valor predictivo negativo de 93,3 %. La detección de tuberculosis multidrogorresistente mostró valores de 89,9 %, 90,6 %, 78,5 % y 95,9 % para sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo, respectivamente. Conclusiones. El análisis de curvas de melting mostró ser seguro y confiable para ser utilizado en el diagnóstico rápido de tuberculosis multidrogorresistente en muestras de esputo.

ABSTRACT Objectives. To analyze melting curves for the diagnosis of multidrug-resistant tuberculosis from sputum samples. Materials and Methods. Sputum samples (n = 250) were collected from patients with clinical suspicion of pulmonary tuberculosis as a result of bacilloscopy and cultured in solid medium Lowenstein Jensen. According to the reference method, 124 samples sensitive to rifampicin and isoniazid, 24 resistant to rifampicin, 33 resistant to isoniazid, and 69 multidrug-resistant were used. It was evaluated by real-time PCR and then by melting curves, the rpoB gene was used as a biomarker of rifampicin resistance, and the katG gene and inhA promoter region were used as biomarkers of isoniazid resistance. The H37Rv strain was considered a drug-sensitive control. The results of the reference method and the results of the melting curve analysis were compared to evaluate the parameters of sensitivity, specificity, positive predictive value and negative predictive value. Results. Rifampicin resistance showed a sensitivity of 90.3%, specificity of 90.4%, positive predictive value of 84.8% and negative predictive value of 94.0%. Isoniazid resistance showed a sensitivity of 90.2%, specificity of 93.9%, positive predictive value of 91.1% and negative predictive value of 93.3%. The detection of multidrug-resistant tuberculosis showed values of 89.9%, 90.6%, 78.5% and 95.9% for sensitivity, specificity, positive predictive value and negative predictive value, respectively. Conclusions. The melting curve analysis showed to be safe and reliable to be used in the rapid diagnosis of multidrug-resistant tuberculosis in sputum samples.

Aplicabilidade da técnica de PCR em tempo real para caracterização de espécies de Leishmania / Aplicability of real time PCR technique for leishmania species characterization

A Leishmaniose Tegumentar Americana (LTA) é causada por protozoários, do gênero Leishmania, envolvidos em um complexo ciclo biológico. No Brasil, sete espécies estão envolvidas com a etiologia da doença, distribuídas em todas as regiões geográficas e responsáveis por diferentes manifestações clínicas. Diante disso, o diagnóstico em conjunto com a identificação da espécie é de grande importância clínico-terapêutica. Este trabalho tem por objetivo avaliar a aplicabilidade da técnica de PCR quantitativa em tempo real (qPCR) para identificação de espécies de Leishmania envolvidas com a etiologia da LTA. Foram realizados ensaios de qPCR para padronização da Temperatura de melting (Tm) utilizando cepas de referência de diferentes espécies de Leishmania. Após o diagnóstico em amostras de sangue de animais domésticos utilizando a qPCR, as amostras positivas foram analisadas através de suas Tm, e os produtos de qPCR foram purificados e sequenciados. Dez amostras previamente caracterizadas por isoenzimas, também foram analisadas através da Tm. Ainda como teste de referência, foi padronizada uma Restriction Fragment Length Polymorphism (RFLP) utilizando as cepas de referência e testada nas amostras. Através da padronização da Tm das espécies, foram criados dois intervalos de análise: 1 (Tm = 78-79,99°C), que compreende: Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis e Leishmania (V.) shawi; e 2 (Tm = 80-82,2°C), que compreende: Leishmania (V.) naiffi, Leishmania (L.) amazonensis e Leishmania (L.) mexicana. Um total de 223 amostras positivas foi analisado, destas, 58 incluídas no intervalo 1 e 165 no intervalo 2. O sequencimento de 94 destas amostras foi correspondente à L. (V.) braziliensis, L. (V.) panamensis e L. (V.) guyanensis. A RFLP em 173 amostras identificou 167 L. (V.) braziliensis, 05 L. (L.) mexicana e 01 L. (V.) panamensis...

The American Cutaneous Leishmaniasis (ACL) is caused by protozoa of the genus Leishmania, involved in a complex biological cycle. In Brazil, seven species are involved in the etiology of the disease, distributed in all geographic regions and responsible for different clinical manifestations. Therefore, the diagnosis together with the identification of the species is of great clinical and therapeutic importance. This work aims to evaluate the applicability of the technique of real-time quantitative PCR (qPCR) for the identification of Leishmania species involved in the etiology of ACL. qPCR assays for standardizing the melting temperature (Tm) using reference strains of different species of Leishmania were performed. After the diagnosis on blood samples of domestic animals using the qPCR positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten samples previously characterized by isoenzymes were also analyzed by Tm. Also as a reference test was standardized as Restriction Fragment Length Polymorphism (RFLP) using the reference strains and tested on samples. Through standardization of Tm species two ranges of analysis were created: 1 (Tm = 78-79,99°C), comprising: Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis e Leishmania (V.) shawi; and, 2 (Tm = 80-82,2°C), comprising: Leishmania (V.) naiffi, Leishmania (L.) amazonensis e Leishmania (L.) mexicana. A total of 223 positive samples were analyzed, of these, 58 included in the range 1 and 165 in the range 2 to 94 and the sequence of these samples corresponded to L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis. By RFLP in 173 samples were identified 167 L. (V.) braziliensis, 05 L. (L.) mexicana and 01 L. (V.) panamensis The analysis of Tm of the ten samples characterized by isoenzymes showed 80% agreement (p = 0.6499) between the gold standard (isoenzymes) and intervals developed in this study...

High resolution melting and its application in identity study of traditional Chinese medicine / 药学学报

High resolution melting (HRM), based on melting curve analysis, requires not only saturating dyes that fluoresce in the presence of double-stranded DNA, but also higher resolution detection equipment. The melting curve is a novel method for sequence matching, genotyping and mutation scanning. The technology is simple, accurate, rapid, closed-tube, low-cost, and high-throughput, which make it gain more and more applications. This review article presents the basic principles, key factors and both the advantage and limitations of HRM. The potential application is discussed in the study of molecular identity of traditional Chinese medicine.

Effect of Tiantai No.1 on gene expression profiles in hippocampus of Alzheimer's disease rats by bioinformatic analysis / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effect of Tiantai No. 1 [symbol in text] on gene expression profile in hippocampus of Alzheimer's disease (AD) rat, molecular genetic target points of the effect of this drug were defined, its molecular genetic pharmacodynamic mechanism of anti-AD was further explored at molecular gene level, and a scientific basis was provided for its clinical availability and promotion.</p><p><b>METHODS</b>Thirty male Sprague-Dawley rats were divided into three groups with 10 rats per group: sham-operation group, model group and Tiantai No. 1 group. Sterile surgical procedure was applied, the model group with bilateral hippocampal injection of Aβ1-40 was established, and normal saline was used instead of Aβ1-40 in the sham-operation group. One week after the models was made, rats were administered by gastric lavage once every day for three consecutive weeks. The rats of the sham-operation group and the model group were daily fed with purified water by lavage; the rats of the Tiantai No.1 group treated group were administered with Tiantai No.1 by lavage. Total RNAs of hippocampus tissues were extracted with Trizol, the changes of hippocampus gene expression profiles in the above three groups were analyzed by using Affymetrix rat whole genome expression profile microarray.</p><p><b>RESULTS</b>Microarray analysis showed that, compared with the sham-operation group, the hippocampus of the model group had 50 up-regulated genes with significant difference (fold change >2), and 21 down-regulated genes with significant difference (fold change <0.5); compared with the hippocampus of the model group, the hippocampus of the Tiantai No. 1 group was found to have 5 up-regulated genes with significant difference (fold change >2) and 20 down-regulated genes with significant difference (fold change <0.5). The functions of differentially expressed genes of the groups were involved in nervous system's development, neuronic differentiation and function-regulation, cellular growth and differentiation and apoptosis, synaptic occurrence and plasticity, inflammation and immune response, ion channels/transporters, cellular signal transduction, cellular material/energy metabolism and so on.</p><p><b>CONCLUSION</b>Tiantai No. 1 can regulate hippocampal function, and further regulate the brain function of animals in multiple gene target points by a number of ways.</p>

Anti-inflammatory effect of Qingwen Baidu Decoction (清瘟败毒饮) in sepsis rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the pharmacological anti-inflammatory mechanism of Chinese formula Qingwen Baidu Decoction (清瘟败毒饮, QBD) from the view of holistic biology.</p><p><b>METHODS</b>The rats were randomly divided into a normal conrol group, a lipopolysaccharide (LPS) group, the low- and high-dose QBD groups, and a dexamethasone (DXM) group. NR8383 cells were treated with culture fluid containing 6% serum from rats of each group respectively. Inflammatory mediators were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blotting hybridization, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) gene array and antibody array.</p><p><b>RESULTS</b>It is showed that the levels of interleukin (IL)-1α, IL-4 and IL-12 were enhanced in the low-dose QBD group; levels of IL-1α, IL-12 and IL-18 were augmented in the high-dose QBD group, compared with the LPS group after ELISA detection. Western blot showed that IL-1β and tumor necrosis factor (TNF)-α expression of the control group were lower than other groups. IL-1β level of the low-dose and high-dose QBD groups detected by RT-PCR was higher in early stage but lower after 24 h than that of the control group (P<0.01). Expression of 84 main inflammatory cytokines and receptors was detected by rat inflammatory cytokines and receptors PCR array. Up-regulation genes were 22 in both the LPS group and the low-dose QBD group, among which 16 up-regulating genes were the same. In these 16 genes, the up-regulating amplitude of 9 genes in the low-dose QBD group was less than that in the LPS group, 4 were similar to and 3 were more. Twenty-nine main cytokines were inspected by rat cytokine antibody array. Intergroup gray value differences were found in 7 expressed cytokines. The levels of these 7 cytokines in the low-dose QBD group were all lower than those in the the LPS group.</p><p><b>CONCLUSIONS</b>QBD has anti-inflammatory effect on sepsis by changing the level of inflammatory mediators.</p>

Characterization of denaturation and renaturation of DNA for DNA hybridization

OBJECTIVES: The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. METHODS: A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. RESULTS: Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. CONCLUSIONS: Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.

One-step methylation variable position analysis technology in single-tube / 法医学杂志

OBJECTIVE@#To develop the single-tube one-step methylation variable position (MVP) analysis technology-single-tube post-digestion PCR-melting curve analysis (PDP-MCA).@*METHODS@#Based on differentially methylated region (DMR) reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme (MSRE), DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples (peripheral venous blood, semen, and vaginal fluid, 5 samples each type) were tested by single-tube one step MVP and traditional MSRE-PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared.@*RESULTS@#When the melting temperature of the fragments had a differential of 2 degrees C, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions (digestion, amplification and detection) into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized.@*CONCLUSION@#Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.

Utilization of high-resolution melting analysis to screen patients with neonatal intrahepatic cholestasis caused by citrin deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).</p><p><b>METHODS</b>Based on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing.</p><p><b>RESULTS</b>HRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing.</p><p><b>CONCLUSION</b>HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.</p>

Detection of exon 7 mutations of PAH gene in classical phenylketonuria by high-resolution melting analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a simple, rapid, inexpensive and sensitive method for detecting hot region for mutations in exon 7 of PAH gene.</p><p><b>METHODS</b>High-resolution melting (HRM) technology was used to detect a c.728G>A mutation in exon 7 in 88 patients with classical type phenylketonuria. Suspected mutations were validated by direct DNA sequencing.</p><p><b>RESULTS</b>The results detected by HRM are in good agreement with the results obtained by direct sequencing.</p><p><b>CONCLUSION</b>HRM analysis is a simple, rapid, inexpensive and sensitive method for detecting hot mutational region in exon 7 of PAH gene.</p>

Detection of Vancomycin-resistant Enterococci using Multiplex Real-time PCR Assay and Melting Curve Analysis / 대한진단검사의학회지

BACKGROUND: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. METHODS: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. RESULTS: Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. CONCLUSIONS: VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis.

Evaluation of DNA melting curve analysis real-time PCR for detection and differentiation of cryptosporidium species

Cryptosporidium is a leading cause of persistent diarrhea in developing countries where it is reported to be more common in malnourished children with more severe consequences of the disease than in well nourished ones. PCR which is the most sensitive and specific diagnostic method for detecting and genotyping Cryptosporidium still has some drawbacks. The present study aimed at evaluating melting curve analysis after real-time PCR in diagnosis and genotyping Cryptosporidium as an alternative to traditional multiplex PCR followed by gel electrophoresis. Seventeen naturally infected immun°Compromised patients suffering from Cryptosporidium diagnosed by both microscopy and immun°Chrommatographic strip assay provided the study isolates. All samples were subjected to traditional multiplex PCR followed by gel electrophoresis and real-time PCR followed by melting curve analysis. Melting curve analysis real-time PCR proved 100% sensitivity and specificity when compared to traditional multiplex PCR. It had the advantage of being less time consuming [1 hr], less liable for post amplification contamination by carry-over as the pr°Cess is completed in a closed tube. Genotyping of Cryptosporidium on the basis of melting curve analysis revealed that C. hominis showed two distinct peaks at 85.5°C and 88.5°C while C. parvum showed two distinct peaks at 80.1°C and 88.5°C. Interestingly, one isolate proved to be a mixed infection showing three peaks at 80.1°C, 85.5°C and 88.5°C. DNA melting curve analysis real-time PCR offers a step forward in the detection and differentiation of Cryptosporidium spp. by circumventing disadvantage of traditional PCR

Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay.

Genomic variations in HIV-1 represent a major problem in understanding disease progression, studying drug resistance and developing effective vaccines. Heteroduplex Mobility Assay (HMA) was used for analyzing HIV-1 subtypes resulting from genetic similarity or divergence of C2 -V3 -V5 region of envelope gene between HIV-1 strains obtained from clinical samples in a tertiary care center at Pune. DNA from the PBMCs of infected individuals was amplified by nested PCR. Heteroduplexes were then formed by denaturing DNA from the unknowns with DNA from the reference strains. The results were analyzed by polyacrylamide gel electrophoresis. Out of 177 samples analyzed, 170 were of subtype C (96%). Four samples were found to be of subtype B (2.2%); in three samples, no definitive assignment of subtype was possible by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. These findings may have significant implications toward development of a candidate vaccine for India.

Rapid detection of common ATP7B mutations in Wilson disease by high resolution melting analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the most prevalent mutations, R778L and P992L of ATP8B gene, in Chinese Wilson disease(WD) patients by high resolution melting (HRM) analysis after polymerase chain reaction (PCR).</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples obtained from 30 cases of WD by the standard phenol/chloroform method. DNA fragments encompassing ATP7B exons 8 and 13 were produced by PCR amplification. The amplicons containing the R778L or P992L mutations were then generated by nested PCR. The nested PCR products were subjected to HRM analysis using the HR-1 instrument. Mutations detected in HRM analysis were verified by restriction analysis using restriction enzyme (MspI or AluI or AfaI) or DNA sequencing.</p><p><b>RESULTS</b>HRM analysis of the fragments encompassing ATP7B exon 8 showed four curve patterns. Subsequent restriction analysis and DNA sequencing proved that the four different curves represent four different genotypes: the wild type, the R778L/R778L homozygote, the R778L heterozygote, and the R778L/752.33delG compound heterozygote. Three HRM curve patterns were observed for the fragments encompassing ATP7B exon 13, representing the wild type, the P992L heterozygote, and the P992L/S975Y compound heterozygote. In our studied samples, allele frequencies of the R778L, P992L and S975Y mutations were 25%, 15% and 1.67%, respectively.</p><p><b>CONCLUSION</b>HRM analysis is a simple, accurate and sensitive approach for rapid detection of the ATP7B mutations and could be used as an optimized method for genetic testing in WD.</p>

Detection of methylation in hepatocellular carcinoma using SYBR Green fluorescent quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.</p><p><b>METHODS</b>Following bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.</p><p><b>RESULTS</b>The amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.</p><p><b>CONCLUSION</b>SYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.</p>

Sperm nuclear histone H2B: correlation with sperm DNA denaturation and DNA stainability / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To examine the relationship between sperm DNA damage and sperm nuclear histone (H2B) staining.</p><p><b>METHODS</b>We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone (H2B) staining and sperm chromatin integrity (assessed by sperm chromatin structure assay and expressed using the percentage of (i) DNA fragmentation index [% DFI] and (ii) high DNA stainability [% HDS)]) were evaluated.</p><p><b>RESULTS</b>Histone H2B immunocytochemistry demonstrated two nuclear staining patterns: (i) focal punctate staining; and (ii) diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men (7.7% +/- 4.6% vs. 1.6% +/- 1.2%, respectively, P < 0.01). We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm % DFI (r = 0.63, P < 0.01) and sperm %HDS (r = 0.63, P < 0.01).</p><p><b>CONCLUSION</b>The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.</p>

Usefulness of Multiplex Real-Time PCR and Melting Curve Analysis in Identification of Nontuberculous Mycobacteria / 대한진단검사의학회지

BACKGROUND: Nontuberculous mycobacteria (NTM) should be correctly identified to the species level, because of different treatment plans among NTM species. This study was performed to assess the usefulness of real-time PCR and melting curve analysis in the identification of NTM. METHODS: One hundred fifty-two clinical NTM isolates were identified to the species level by PCR-restriction fragment length polymorphism analysis (PRA). Those strains were then identified by multiplex real-time PCR and melting curve analysis on the 16S rRNA gene and hsp65 gene. RESULTS: In the 16S rRNA gene fragment analysis, M. abscessus-M. chelonae group showed melting point at temperatures above 65 degrees C and M. avium complex (MAC; M. avium and M. intracelluare) below 48 degrees C, which differentiated M. abscessus-M. chelonae group and MAC from other NTM. In the hsp65 gene fragment analysis, M. abscessus-M. chelonae group was clearly divided into M. abscessus type I, M. abscessus type II, and M. chelonae according to the melting points at 61.25 degrees C, 66.06 degrees C, and 57.58 degrees C, respectively. CONCLUSIONS: With the multiplex real-time PCR and melting curve analysis of 16S rRNA and hsp65 genes, M. abscessus and M. chelonae were readily identified and MAC were differentiated from other NTM. Especially, M. abscessus and M. chelonae, which were not differentiated from each other with the 16S rRNA gene fragment analysis, were identified with hsp65 gene fragment analysis.

Mutation detection of PKD2 gene in Chinese by denaturing high-performance liquid chromatograph / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the mutations of autosomal dominant polycystic kidney disease gene 2(PKD2)in Chinese.</p><p><b>METHODS</b>The white blood cell genomic DNA from patients of 94 Chinese autosomal dominant polycystic kidney disease(ADPKD) pedigrees was isolated and amplified by polymerase chain reaction(PCR). The PCR products were analyzed by denaturing high-performance liquid chromatography(DHPLC). The samples with abnormal profiles were sequenced.</p><p><b>RESULTS</b>Eight mutations were identified, including 2 nonsense mutations, 2 deletion mutations,1 insertion mutation and 3 missense mutations. Two nonsense mutations occurred in exon 5(1249C-->T) and exon 13(2407C-->T),both resulted in a stop codon. The insertion was in exon 2(636-637 ins T),and the deletion mutations were in exons 12(2348-2351 del AGAA) and 13(2401 delete A),resulting in the reading frame shift. Three missense mutations were in exons 1(G568-->A),4(C964-->T),and 5(G1168-->A), which caused amino acid changes (190Ala-->Thr,322Arg-->Trp,390Gly-->Ser).</p><p><b>CONCLUSION</b>The method of DHPLC was used in detecting mutations successfully and 8 mutations in PKD2 were identified. It will be useful in the molecular diagnosis of ADPKD in advance of the cysts formation and birth.</p>

Una especie nueva de Dennstaedtia (Filicales: Dennstaedtiaceae) para México / New species of Dennstaedtia (Filicales: Dennstaedtiaceae) to Mexico

Dennstaedtia gracilis A. Rojas et Tejero (Dennstaedtiaceae) is herein described and illustrated as a new species endemic to Mexico. Its belongs to the group of winged adaxial secondary costae species but it differs by the combination of characters as smaller fronds, deltate pinnules and lobed segment apex

Detección y genotipificación del papillomavirus humano (HPV) por PCR-LIS-SSCP / Detection and genotyping of human papillomavirus (HPV) using PCR-LIS-SSCP

This study describes a fast and simple method for human papillomavirus (HPV) typing based on the polymerase chain reaction (PCR) amplification of a portion of the viral genome and single strand conformation polymorphism using low ionic strength solutions (LIS-SSCP). PCR products were obtained using My09/My11 and Gp5/Gp6 primers in a nested reaction. The band patterns corresponded to the plasmid HPV clones from HPV-6, -11, -16, -18, -31, -33 and -34. The SSCP minigels were stained with SYBR-Green II. In order to determine diagnostic applicability, 100 cervical samples were studied comprising liquid cytology and paraffin embedded biopsies from patients showing squamous intraepithelial lesions (SILs). The SSCP patterns obtained from the clinical samples and the HPV clones were similar when the same type was present. Therefore, the methodology proved to be efficient and with high reproducibility for the detection and typing of HPV in clinical samples.

Detection of point mutations by non-isotopic single strand conformation polymorphism

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens