HPV detection in oral mucosa samples in pediatric patients / Detecção de HPV em amostras de mucosa oral em pacientes pediátricos

ABSTRACT INTRODUCTION: The human papillomavirus (HPV) detection favors treatments for patients with clinical manifestations and limits future consequences for those with asymptomatic infections. OBJECTIVES: Therefore, the present study aimed to evaluate the sensitivity of polymerase chain reaction (PCR) for HPV detection from oral mucosa samples, of asymptomatic patients and patients with clinical manifestations of laryngeal papillomatosis. MATERIAL AND METHODS: A total of 49 pediatric patient samples were obtained by exfoliation of the oral mucosa with a sterile brush. The deoxyribonucleic acid (DNA) samples was extracted and used for HPV detection, using GP5 and GP6 oligonucleotides, by conventional PCR and qPCR reactions. RESULTS: Among the 49 samples, eight were from patients clinically diagnosed with laryngeal papillomatosis, but in both conventional PCR and qPCR technic, only one sample had presented positivity. DISCUSSION: These results suggest that the sample type, the methodology used to collect, the extraction methodology used, the anatomical location of the lesion and the oligonucleotides used; all factors strongly influence the sensitivity of HPV detection by PCR methodology. CONCLUSION: Thus, more studies are needed to better determine the sample collection, and the processing techniques present more reproducibility on PCR detection.

RESUMEN INTRODUCCIÓN: El virus del papiloma humano (VPH) ayuda los tratamientos de pacientes que presentan manifestaciones clínicas y limita las consecuencias futuras para aquellos con infecciones asintomáticas. OBJETIVOS: Evaluar la sensibilidad de la reacción en cadena de la polimerasa (PCR) para detectar VPH en diferentes muestras. MATERIAL Y MÉTODOS: Cuarenta y nueve muestras de pacientes pediátricos se obtuvieron por exfoliación de la mucosa oral con un cepillo estéril. Se utilizó el ácido desoxirribonucleico (ADN) de esas muestras para detectar VPH por PCR convencional y PCR cuantitativa en tiempo real (qPCR). RESULTADOS: Entre las 49 muestras, ocho eran de pacientes clínicamente diagnosticados con papilomatosis laríngea; sin embargo, tanto en la PCR convencional como en la qPCR, sólo una muestra presentó amplificación del fragmento esperado. DISCUSIÓN: Eses resultados sugieren que el tipo de muestra, el método empleado en la recolección, el método de extracción, la ubicación anatómica de la lesión y los oligonucleótidos utilizados influyen fuertemente la sensibilidad de detección de VPH por PCR. CONCLUSIÓN: Se necesita mayor investigación para determinar las mejores técnicas de recolección y procesamiento de muestras para que la detección de VPH por PCR sea más eficiente.

RESUMO INTRODUÇÃO: A detecção do papilomavírus humano (HPV) auxilia os tratamentos para pacientes que apresentam manifestações clínicas e limita as consequências futuras para os que apresentam infecções assintomáticas. OBJETIVOS: Avaliar a sensibilidade da reação em cadeia da polimerase (PCR) para detecção de HPV em diferentes amostras. MATERIAL E MÉTODOS: Quarenta e nove amostras de pacientes pediátricos foram obtidas por esfoliação da mucosa oral com uma escova estéril. O ácido desoxirribonucleico (DNA) dessas amostras foi utilizado para detecção de HPV por PCR convencional e PCR em tempo real (qPCR). RESULTADOS: Das 49 amostras, oito eram de pacientes clinicamente diagnosticados com papilomatose laríngea; porém, tanto na PCR convencional quanto na qPCR, apenas uma amostra apresentou amplificação do fragmento esperado. DISCUSSÃO: Esses resultados sugerem que o tipo de amostra, a metodologia empregada na coleta, a metodologia de extração empregada, a localização anatômica da lesão e os oligonucleotídeos utilizados influenciam fortemente a sensibilidade da detecção de HPV por PCR. CONCLUSÃO: Mais estudos são necessários para determinar as melhores técnicas de coleta e processamento das amostras a fim de que a detecção de HPV por PCR seja mais eficiente.

A improved tetra-primer amplification refractory mutation system PCR based assay to detect four single nucleotide polymorphisms associated with folate metabolism in a single reaction / 中华检验医学杂志

Objective To develop a tetra-primer amplification refractory mutation system PCR(T-ARMS-PCR)assay for detecting four single nucleotide polymorphisms(SNPs): rs1801133,rs1801131, rs1805087 and rs1801394 associated with folate metabolism in a single reaction tube.Methods Methodology was developed.Then, it applied the established method to analyze 150 physical examination children′s anticoagulant blood samples admitted to the Department of Clinical Laboratory, Children′s Hospital of Hebei Province from January 2017 to April 2017.Four sets of chimeric primers consisting of a universal Tag sequence and targeting rs 1801133,rs1801131,rs1805087 and rs1801394 fused to the specific sequence were designed according to the T-ARMS-PCR principle and the multiplex chimeric primers strategy.A single tube PCR was then conducted after optimizing the primer concentrations and the reaction conditions.The amplified products were analyzed by QIAxcel capillary electrophoresis, and the corresponding SNP genotypes of 150 samples were identified.Furthermore,all samples were verified by direct sequencing.And the Hardy-Weinberg Equilibrium(HWE)testing of four SNPs from 150 samples were conducted by SPSS17.0 Chi-square test.Results The improved T-ARMS-PCR combined with capillary electrophoresis can accurately verify eight different alleles of the four SNPs associated with folate metabolism at one time in 3 hours.Four SNPs of 150 whole blood samples were accurately classified and the results were completely consistent with direct sequencing.All the genotype frequencies of these four SNPs were in HWE (χ2rs1801133=0.69, Prs1801133=0.40; χ2rs1801131=0.21, Prs1801131=0.64; χ2rs1805087=3.32, Prs1805087=0.07;χ2rs1801394=1.91, Prs1801394=0.17).Conclusions The proposed improved T-ARMS-PCR combined with capillary electrophoresis in this study can accurately verify four SNPs associated with folate metabolism in a single reaction tube.This method might be a valuable tool to specifically guide the folate supplement in general population.

Identification of novel HLA alleles：HLA-A*24:233 and HLA-A*26:89 / 中国组织工程研究

BACKGROUND:The discovery of novel HLA aleles is accelerated by development of molecular biology and applications of numerous new technologies. These new findings not only rich HLA family, but also find a breakthrough point for the study of genetic superiority or disappearance of national gene. OBJECTIVE:To identify two novel HLA-A aleles and to analysis their nucleotide sequences. METHODS:The two samples from two volunteers of Chinese Marrow Donor Program were detected using PCR-SBT and GSSP methods. The HLA-A locus in the two samples were both abnormal genes, and the nucleotide sequence differences were analyzed. RESULTS AND CONCLUSION: The sequences of the samples were different from al aleles in the HLA databases. Sample 1 was found to be different from the closet matching alele A*24:02:01 in one nucleotide substitutions, 360 G > C in exon 3, resulting in amino acid changed from glutamine to histidine at codon 96; and sample 2 differed from A*26:01:01 in one nucleotide substitution, 97 T > C in exon 2, resulting in amino acid changed from tyrosine to histidine at codon 9. The novel aleles were identified and assigned the name HLA-A*24:233 and HLA-A*26:89 officialy by the WHO Nomenclature Committee.

Comparison of different fluorescent quantitation polymerase chain reaction for genes detection of Streptococcus pneumonia / 中国医师进修杂志

Objective To compare the sensitivity and specificity of different fluorescent quantitation polymerase chain reaction(PCR) for genes detection of Streptococcus pneumonia.Methods By designing five sets of fluorescent quantitation PCR primers targeting different genes of Streptococcus pneumonia (lytA,ply,spn9802,psaA,cspA),the sensitivity and specificity of fluorescent quantitation PCR was determined through detecting 37 strains of Streptococcus pneumonia and 28 strains of non-Streptococcus pneumonia,as well as 80 sputum specimens from suspected pneumonia cases.Results These five primers had excellent sensitivity with 90％ amplification efficiency through analysis of the standard curve.The change of Ct value was less than 0.5,indicating that these five primers have good stability.By detecting 37 strains of Streptococcus pneumonia and 28 strains of non-Streptococcus pneumonia,it showed that the specificity of lytA,ply and spn9802 was better than the other two primers (positive rate and negative rate was all 100％).But the detection limit of lytA,ply was one order of magnitude than spn9802 (101 CFU/ml vs.102 CFU/ml).The specificity of psaA and cspA was worse [negative rate of psaA was 89％ (25/28),positive rate of cspA was 97％ (36/37)].By detecting 80 sputum specimens from suspected pneumonia cases,it showed that the specificity of fluorescent quantitation PCR were better than the bacterial culture.The specificity of lytA and ply was the best two [positive rate:92.50％(74/80),90.00％(72/80)],and next was spn9802 [86.25％(69/80)],the specificity ofpsaA and cspA was the worst two for detection [76.25％(61/80) and 78.75％(63/80)].Conclusions The sensitivity and specificity of fluorescent quantitation PCR genes used in clinic for detecting Streptococcus pneumonia is different.LytA and ply are the best primers in specificity,next is spn9802,and psaA and cspA are the worst primers for in specificity detection.

Establishment and application of DPO-based quadruple real-time PCR for detection of mycobacteria / 中华检验医学杂志

Objective To establish a rapid,accurate and specific method to detect the common mycobacteria based on multiplex real-time PCR.Methods The dual priming oligonucleotide ( DPO)primers and TaqMan probes labeled with FAM,ROX,HEX or JOE fluoresceins at 5' end and eclipse at 3' end respectively were designed to detect the 16S rRNA of mycobacteria.Both specificity and sensitivity were estimated on multiplex real-time PCR detecting genome DNA from 4 mycobacterial model species.Sixty eight early morning sputum specimens collected from hospitalized patients in the Red Cross Hospital of Hangzhou were detected by multiplex real-time PCR,bacterial culture and smear microscopy simultaneously.The positive rates were analyzed by chi-square.Results Mycobacteria including Mycobacterium tuberculosis and three common non-tuberculosis mycobacteria spp.were identified by multiplex real-time PCR accurately and specifically,with the limited load at 101 cfu/ml.In 68 sputum specimens,31 were positive (positive rate 45.6％ ) by this method,which was significant higher than that by smear microscopy ( positive rate 14.7％,x2 =15.4,P ＜0.05 ).The positive cases were identified as 28 Mycobacterium tuberculosis,1 Mycobacterium avium and 2 Mycobacterium intracellulare in agreement with the culture results.One case,which is detected by culture,but not by PCR,was identified as Mycobacterium chelonae by sequencing.Conclusion The multiplex real-time PCR characterizing as sensitive,specific and time-saving for Mycobacterium tuberculosis and common non-tuberculosis mycobacteria could be chosen as the rapid laboratory test of mycobacterial infection.

Comparison of different primes for PCR-based diagnosis of cutaneous leishmaniasis

OBJECTIVE: The objective of this study was to analyze different primers that are commonly used in epidemiological studies for the detection of Leishmania DNA by PCR, and to compare them to the conventional direct parasite search for American cutaneous leishmaniasis (ACL) diagnosis. MATERIAL AND METHODS: Five pairs of primers, four of them derived from Leishmania kDNA sequences (MP3H-MP1L; B1-B2; LBF1-LBR1; 13A-13B), and one derived from the SL RNA (mini-exon) gene repeat (LU5A-LB3C), reported previously, were used. RESULTS: The MP3H-MP1L primers were the best at amplifying the DNA, detecting 2 fg of Leishmania spp. DNA. The 13A-13B primers presented the worst performance, detecting 512 x 10³ fg of DNA. CONCLUSION: The wide variation in the analytical sensitivity of the primers used in the PCR, and the significant differences from the conventional method of ACL diagnosis found in this study, emphasize the importance of standardizing the PCR technique, analyzing sensitivity, and selecting suitable oligonucleotide primers.

Painel molecular para detecção de microrganismos associados à sepse / Molecular panel for detection of sepsis-related microorganisms

INTRODUÇÃO: A sepse é uma resposta inflamatória sistêmica relacionada com altas taxas de mortalidade no meio hospitalar. O diagnóstico etiológico tardio e terapia antimicrobiana inadequada se associam a falhas do tratamento. Exames moleculares baseados na reação em cadeia da polimerase são considerados métodos mais rápidos e precisos do que técnicas de hemocultura para identificação microbiana, proporcionando uma taxa mais elevada de sucesso terapêutico. OBJETIVO: Desenvolver um painel de seqüências iniciadoras (primers) para fragmentos de DNA de microrganismos associados à sepse. MÉTODOS: Seqüências iniciadoras para amplificação de Enterobacter spp., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus e Candida spp. foram desenvolvidos e testados quanto a sensibilidade e especificidade com base em suas respectivas cepas padrão. RESULTADOS: A especificidade pretendida foi obtida para os primers de P. aeruginosa, S. aureus e Candida spp. O teste de sensibilidade mostrou um limite de detecção de 5 ng a 500 fg em amostras de sangue contaminado com DNA microbiano. CONCLUSÕES: O painel molecular apresentado oferece a vantagem de constituir um sistema flexível "aberto" em comparação a outros métodos de detecção múltipla.

INTRODUCTION: Sepsis is a systemic inflammatory response related to high mortality rates in the hospital environment. Delayed etiological diagnosis and inadequate antimicrobial therapy are associated with treatment failures. Molecular tests based on polymerase chain reaction are regarded as faster and more accurate procedures than culture techniques for microbial identification, providing a higher rate of therapeutic success. OBJECTIVE: To develop a panel of primers for DNA fragments of sepsis-related microorganisms. METHODS: Primers for amplification of Enterobacter spp., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp. were designed and tested for sensitivity and specificity on the basis of their respective standard strains. RESULTS: The intended specificity was obtained for P. aeruginosa, S. aureus and Candida spp primers. Sensitivity tests showed a threshold for detection from 5 ng to 500 fg in blood samples contaminated with microbial DNA. CONCLUSIONS: The molecular panel presented offers the advantage of a flexible 'open' system when compared to other multiplex detection methods.

Increasing specificity of real time PCR to detect microRNA through primer design and annealing temperature increase / 北京大学学报(医学版)

Objective: To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA ( miRNA) ,and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs. Methods: Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini. Results: Raising annealing temperatures 12℃-14℃above the T_m of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels. Conclusion: Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.

The relationship between single nucleotide polymorphism in the coding region of class Ⅱ transactivator gene and outcomes of chronic hepatitis B virus infection / 中华传染病杂志

Objective To investigate the relationship between the non-homonymy single nucleotide polymorphism(SNP)of C19170G,C30799G in the coding area of class Ⅱ transaetivator(CII TA)and the different clinical phenotypes of chronic hepatitis B virus(HBV)infection.Methods Six hundred and twenty-seven patients with chronic HBV infection and 101 healthy blood donors were enrolled in this study.Genotyping of C19170G,C30799G in C Ⅱ TA gene coding region were done by sequence-specific primer polymerase chain reaction(PCR-SSP).Hardy-Weinberg balance of the genotypes was analyzed using chi-square test.Differences between two sets were tested by contingency table chi-square test and unconditional Logistic regression was performed. Results The frequencies of G allele and GG+GC genetypes at 19170 site were significantly higher in patients with liver cirrhosis than those with non-progressive liver diseases(X2=7.128,P=0.008;X2=6.404,P=0.011,respectively).There were significantly differences of the allele frequencies between patients with liver cirrhosis and non-progressive liver diseases(OR:0.742,95%CI:0.552～0.998,P=0.048),and the main differences were observed in G dominant model(OR:0.581,95% CI:0.353～0.954,P=0.032).The frequencies of C allele and CC genotype at 30799 site were significantly higher in patients with hepatocellular carcinoma than those in patients with liver cirrhosis(X2=4.861,P=0.027;X2=4.993,P=0.025).There were significant differences of the genotype frequencies at 30799 site between patients with liver cirrhosis and hepatocellular carcinoma(OR:0.557,95% CI:0.334～0.930,P=0.025),and the differences were mainly observed in C recessive model(OR:0.491,95% CI:0.269～0.898,P=0.021).Conclusions The polymorphisms at 19170 site are associated with liver cirrhosis in chronic HBV infection,and the G allele carriers are prone to progress into liver cirrhosis.The polymorphisms at 30799 site are associated with hepatocellular carcinoma in chronic HBV infection,and CC genotype carriers are prone to progress into hepatocellular carcinoma.

Preliminary exploration of the influence factors of degenerate oligonucleotide primered PCR of genome DNA / 北京大学学报(医学版)

Objective: To explore the influence factors of the degenerate oligonucleotide primered PCR(DOP-PCR). Methods: Genome DNA template from the mouse single oocyte or liver tissue were used to perform DOP-PCR. DOP-PCR was carried out with templates of different origin, different gradient dilution, with or without low melting point gel purified to wipe off the small fragment that might interfere with the following analysis, and then PCR of gene FTCD and CBS were carried out to evaluate the influence of these factors on the amplification efficiency and specificity. Results: Compared with genome DNA template from mouse liver, the template from single oocyte had the same efficiency and specificity but a minor yield and different gradient dilution of DNA template had no effect on the efficiency and specificity. Furthermore, there was a higher specificity in the low melting point gel-purified DOP-PCR product than in untreated ones. Conclusion: We have got a satisfactory result and increased specificity from DOP-PCR product purified with the low melting point gel. Single oocyte of mice could be used for further investigation of special genes detection by DOP-PCR and of an optimization in the yield of the products.

Identification of Sporothrix schenckii Based on the Nucleotide Sequences in Internal Transcribed Spacer Region 2 of Ribosomal DNA Genes / 中华皮肤科杂志

Objective To study the molecular identification method of Sporothrix schenckii based on the nucleotide sequences in internal transcribed spacer region 2 of ribosomal DNA (rDNA) genes. Methods Species-specific primers were used to amplify the internal transcribed spacer region 2 of rDNA of 22 clinical isolates of Sporothrix schenckii and 12 strains of dematiaceous fungi. Totally 11 strains of Sporothrix schenckii were sequenced and analyzed, in which 1 strain came from the US and the others were isolated from different areas in China. A pair of species-specific oligonucleotide primers (SSP) were designed based on the ITS2 sequence. With the species-specific primers, rDNA of Sporothrix schenckii and dematiaceous fungi were amplified by PCR. Results Sequencing and analysis showed that internal transcribed spacer region 2 of rDNA gene was conservative in Sporothrix schenckii. A 300 bp fragment was obtained from 22 strains of Sporothrix schenckii, but not from the other species. Conclusions This method is specific, sensitive and reliable for the identification of Sporothrix schenckii and could be used for clinical molecular diagnosis.