The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu sub sequentesequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods: a comparative study / 中国输血杂志

【Objective】 To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe (Flow-rSSO) hybridization and sequencing based typing-PCR (PCR-SBT) methods. 【Methods】 A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method, and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay. The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed. Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number, and further tested using the PCR-SSP commercial kit. 【Results】 The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods. Two samples, one with 3DL1 negative, the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO, were actually all positive tested by PCR-SBT. Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive, which agreed well with PCR-SBT results. 【Conclusion】 In this paper, the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong, which indicated the importance of carrying out the quality control for reagents in KIR gene testing.

DNA molecular identification of Polyrhachis dives medicinal materials / 药学学报

In the study, specific primers were designed based on the CO Ⅰ gene sequence of Polyrhachis dives. By optimizing the genomic DNA extraction method and amplification conditions, we established an efficient, specific, and accurate DNA molecular identification method for Polyrhachis dives. In this method, the length of the target fragment was 294-308 bp, and the other counterfeits had no target bands. In this paper, the specific identification method of the origin of Polyrhachis dives established can be used to identify the medicinal materials of Polyrhachis dives accurately.

Shoot rot of Zizania latifolia and the first record of its pathogen Pantoea ananatis in China / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The aquatic grass Zizania latifolia grows symbiotically with the fungus Ustilago esculenta producing swollen structures called Jiaobai, widely cultivated in China. A new disease of Z. latifolia was found in Zhejiang Province, China. Initial lesions appeared on the leaf sheaths or sometimes on the leaves near the leaf sheaths. The lesions extended along the axis of the leaf shoots and formed long brown to dark brown streaks from the leaf sheath to the leaf, causing sheath rot and death of entire leaves on young plants. The pathogen was isolated and identified as the bacterium Pantoea ananatis, based on 16S ribosomal RNA (rRNA) gene sequencing, multilocus sequence analysis (atpD (β-subunit of ATP synthase F1), gyrB (DNA gyrase subunit B), infB (translation initiation factor 2), and rpoB (β‍-subunit of RNA polymerase) genes), and pathogenicity tests. Ultrastructural observations using scanning electron microscopy revealed that the bacterial cells colonized the vascular tissues in leaf sheaths, forming biofilms on the inner surface of vessel walls, and extended between vessel elements via the perforated plates. To achieve efficient detection and diagnosis of P. ananatis, species-specific primer pairs were designed and validated by testing closely related and unrelated species and diseased tissues of Z. latifolia. This is the first report of bacterial sheath rot disease of Z. latifolia caused by P. ananatis in China.

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.

HLA- A & B Association in Psoriasis in Tamil Speaking South Indian Population.

Psoriasis is an auto-immune skin disease affecting skin, nails and joints. The association of HLA with psoriasis is already established with HLA- C*06 known to be associated strongly with the disease. We wanted to determine the HLA -A & HLA-B pattern and its association with psoriasis in a Tamil speaking ethnic population.METHODSA total of 100 psoriasis patients attending the Dermatology OPD at SRMC were taken up for the study. This was a case control study and hence 100 voluntary blood donors donating at the SRMC Hospital blood bank were taken up for study as controls. Voluntary blood donors are considered as healthy normal individuals and hence chosen as controls. All the 100 patients and 100 controls were typed for HLA (Human Leucocyte Antigen) - A & B by PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primers) and the results were analysed statistically using OpenEpi software (2 X 2 table). The Odds Ratio (OR), p (probability) value, and 95% confidence interval were the statistical tests which were studied.RESULTSHLA-A*02, 24 and HLA-B*35 were found to be strongly associated with psoriasis among Tamil speaking ethnic population.CONCLUSIONSThere are different HLA – A & B alleles associated with psoriasis in Tamil ethnic population in comparison with other ethnic studies

Association of HLA-C 06 with Psoriasis in a Tertiary Care Hospital.

Prevalence of psoriasis is 1-3% in India. HLA-C*06 has been shown to be strongly associated with psoriasis in different ethnic populations. This study was carried out to determine the association of HLA-C in psoriasis patients in a south Indian ethnic population.METHODSA total of 200 samples were included in the study. In all, 100 psoriasis patients and 100 healthy controls were studied. HLA-C typing was done by PCR-SSP method. Results were analysed statistically using open epi software (2 X 2 table). The Odds ratio (OR), p (probability) value, and 95% confidence interval were the statistical tests applied and analysed.RESULTSA total of 14 different HLA-C alleles were identified in both 100 cases and 100 controls. Among the 14 different HLA-C alleles, the alleles which were found to be strongly associated with psoriasis which were statistically significant were both HLA-C*06 and HLA-C*07. HLA-C*06 was found to be present in 52% of the patients and HLA-C*07 was found to be present in 33% of the patients. HLA-C*06 was found to be strongly associated with the disease in 52% of the patients.CONCLUSIONSThis study confirms HLA-C*06 association with psoriasis which is in concordance with other previous studies.

DNA Barcoding Identification of Paecilomyces hepiali and Jinshuibao Capsules / 中国实验方剂学杂志

Objective::Because traditional methods are difficult to identify the fermentation mycelium, DNA barcoding technology was used to quickly identify the raw material strain Paecilomyces hepiali of Jinshuibao capsules and related products. Method::A total of 168 samples of 8 species of P. hepiali and its confusable species were identified by internal transcribed spacer (ITS) sequences, and based on the ITS sequences, P. hepiali specific primers were designed to quickly identify the related products. Result::The length of ITS sequences in 44 P. hepiali samples from different sources was 499 bp and there was no mutation site. It was shown that P. hepiali could be distinguished from 7 confusable species based on ITS sequences. The specific primer (ITS-BF/ITS-BR) of P. hepiali designed by ITS sequences could be amplified to obtain a short fragment of 102 bp in length, which could be used to rapidly identify P. hepiali from other confusable species, and to distinguish relevant products in the market. Conclusion::The rapid identification of P. hepiali and its related products can be achieved through the ITS sequences and specific primers, which provides a reference for the production and quality control of Jinshuibao capsules.

Development and Comparison of Determination of Staphylococcus aureus in Different Processed Products of Angelicae Sinensis Radix Based on Real-time PCR Technique / 中国实验方剂学杂志

Objective:To quantitatively analyze the changes of Staphylococcus aureus in different processed products of Angelicae Sinensis Radix. Method:The real-time fluorescence quantitative polymerase chain reaction method （Real-time PCR） was established to quantitatively analyze S. aureus in Angelicae Sinensis Radix decoction pieces which bought from different producing areas， different enterprises and different storage time. The fluorescence quantitative reaction system was SYBR Premix Ex Taq Ⅱ of 10 μL， each of forward primer and reverse primer （10 μmol·L-1） of 0.8 μL， template/genome DNA of 1 μL， double distilled water of 7.4 μL. The reaction conditions of the fluorescence quantitative amplification curve were pre-denaturing for 30 s at 94 ℃， denaturing for 10 s at 94 ℃， annealing for 12 s at 60 ℃， extensing for 30 s at 72 ℃， cycling 45 times， single-point detection signal at 72 ℃. The melting curve was made from 72 ℃， and the step temperature of 0.5 ℃ was kept for 15 s to collect fluorescence. According to the results of Real-time PCR， representative samples were selected from Angelicae Sinensis Radix decoction pieces for comparison between plate counting method and Real-time PCR. Result:The content of S. aureus in different processed products was sorted by rank of raw Angelicae Sinensis Radix>soil-fried Angelicae Sinensis Radix>wine-processed Angelicae Sinensis Radix. The content of S. aureus was the lowest in the samples from Weiyuan area of Gansu province by comparing with other producing areas. Compared with the retail enterprises， the content of S. aureus in raw products and wine-processed products from production and sale enterprises was lower. Different storage time had certain effect on the content of S. aureus in raw products and wine-processed products， and the content of S. aureus increased with the increase of storage time. The detection results of plate counting method were 3-4 orders of magnitude lower than that of Real-time PCR. Conclusion:The established Real-time PCR is superior to plate counting method in specificity， sensitivity， reliability and reporting period， which can provide an effective method for rapid and accurate quantitative detection of S. aureus in different processed products of Angelicae Sinensis Radix.

Utilizing bioinformatics to detect genetic similarities between African honey bee subspecies

Various honey bees, especially subspecies Apis mellifera, occur in Africa and are distribute across the continent. The genetic relationships and identical genetic characteristics between honey bee subspecies in Africa (African bee subspecies) have not been widely investigated using sequence analysis. On the other hand, bioinformatics are developed rapidly and have diverse applications. It is anticipated that bioinformatics can show the genetic relationships and similarities among subspecies. These points have high importance, especially subspecies with identical genetic characteristics can be infected with the same group of pathogens, which have implications on honey bee health. In this study, the mitochondrial DNA sequences of four African subspecies and Africanized bees were subjected to the analyses of base composition, phylogeny, shared gene clusters, enzymatic digestion, and open reading frames. High identical base composition was detected in the studied subspecies, and high identical results from all tests were found between A. m. scutellata and A. m. capensis followed by A. m. intermissa and A. m. monticola. The least genetic relationships were found between A. m. lamarckii and the other subspecies. This study presents insights into the genetic aspects of African bee subspecies and highlights similarity and dissimilarity aspects. Also, Africanized honey bees derived from A. m. scutellata showed high genetic similarities to other African bees, especially A. m.capensis. Additionally, specific primers to identify these subspecies were designed and tested.

Genetic diversity and population structure analysis of Asian and African aromatic rice (Oryza sativa L.) genotypes

Rice germplasms collected from different regions could be used as valuable resources for the future breeding programme. For the utilization of such collections, knowledge about the level and distribution of genetic diversity among these collections will facilitate the breeder. In this study, we report the phenotypic correlation, biochemical quality parameters and population genetic analysis of 35 rice accessions including 34 aromatic rice from different countries and a nonaromatic, Nagina 22, a well-known drought resistance variety. Further biochemical quality analysis, gel consistency test, molecular diversity analysis with 55 simple sequence repeat markers, population structure analysis and pair wise FST analysis were also conducted to assess the genetic diversity. The collected rice genotypes showed significant variability in different agronomic traits, i.e. spikelet per panicle, branches per panicle etc. Results obtained from the above tests demonstrated the importance of regional genetic studies for understanding the diversification of aromatic rice in Asian and African rice.

Translation initiation codon (ATG) or SCoT markers-based polymorphism study within and across various Capsicum accessions: insight from their amplification, cross-transferability and genetic diversity

Being an economical and nutritional crop, Capsicum appeases people’s peppery taste and is found to bewidely distributed all over the world having vast diversity. In the present study, genetic polymorphism, cross transferability (CT) and genetic diversity were examined among the 54 different accessions of Capsicum species including 49 of Capsicum annuum, three of C. baccatum and two of C. frutescens, using a set of 36 start codon targeted (SCoT) primers. Of the total, 35 SCoT markers showed successful amplification profile among chilli germplasms and an average primer polymorphism was reported as 81.52% which ranged from 50% (SCoT-6) to 100% (SCoT-11). A total of 365 amplicons were obtained with an average of 10.43 bands per primer and the length of the bands ranged from 150 bp to 1.2 kb. Further, polymorphic information content value of SCoT markers ranged from 0.42 (for SCoT-25) to 0.86 (SCoT-27) with an average of 0.78. The average value of CT of SCoT markers was 44.08% ranged from 14.25% to 57.26% among different chilli accessions. A dendrogram was constructed and established genetic relationship among 54 capsicum species, with the help of translation initiation codon polymorphisms or SCoT primer amplification. This study suggests the effectiveness of SCoT marker system for characterizing and assessing genetic diversity of Capsicum germplasm, which can be used for evolutionary studies and to identify agronomically important traits.

Molecular Variability of Ocimum gratissimum L. Accessions Using RAPD Marker

Aim: This study investigated the molecular variability among accessions of Ocimum gratissimum from selected states in Nigeria and Mali using RAPD marker. Study Design: The experimental design was complete randomized design (CRD) with three replicates. Materials and Methods: Twenty accessions of Ocimum gratissimum were collected from nineteen selected Local Governments in four South-western States of Nigeria (Ogun, Oyo, Osun and Lagos) and Mali, to assess their genetic diversity and phylogenetic relationship. Molecular statistics of binary data generated from Random Amplified Polymorphic DNA (RAPD) marker was conducted using numerical taxonomic and multivariate analysis (NTSYS-PC) package, while dendrogram was constructed by Jaccard’s similarity coefficient using unweighted paired group method of arithmetic mean (UPGMA). Results: Accession Y3 from Ona-Ara yielded the highest total volume of DNA concentration (736.9 µ/l), while the highest genomic DNA concentration of 2.44 ng/ was recorded in accession L-04 from Agege. Out of total number of 52 bands from three primers of RAPD, 48 produced polymorphic amplified products. OPO-08 primer was highly polymorphic with 94.73%, and had the highest allele numbers, gene diversity and polymorphic information contents of 16.0, 0.914 and 0.909 respectively, while OPO-06 produced the highest number of 20 polymorphic bands. Cluster II was the highest group in the dendogram, and comprised of two states (Oyo and Lagos) and Mali which constituted seven accessions; Y-03 (Ona-Ara), Y-04 (Egbeda), Y-05 (Ido), L-01 (Surulere), L-03 (Ifako-Ijaye), L-04 (Agege) and M (Mali). Accession S-03 from Ife-North was the most distant with highest similarity index of 1.188. Conclusion: The RAPD is highly polymorphic, and could be useful in characterizing and revealing wide range of genomic variation and phylogenetic relationship among different accessions of O. gratissimum with broad genetic base.

Missed detections of influenza A(H1)pdm09 by real-time RT–PCR assay due to haemagglutinin sequence mutation, December 2017 to March 2018, northern Viet Nam

Introduction@#There are two methods of reverse transcription polymerase chain reaction (RT–PCR) that have been the common methods to detect influenza infections: conventional and real-time RT–PCR. From December 2017 to March 2018, several missed diagnoses of influenza A(H1)pdm09 using real-time RT–PCR were reported in northern Viet Nam. This study investigated how these missed detections occurred to determine their effect on the surveillance of influenza.@*Methods@#The haemagglutinin (HA) segments of A(H1N1)pdm09 from both real-time RT-PCR positive and negative samples were isolated and sequenced. The primer and probe sets in the HA gene were checked for mismatches, and phylogenetic analyses were performed to determine the molecular epidemiology of these viruses.@*Results@#There were 86 positive influenza A samples; 32 were A(H1)pdm09 positive by conventional RT–PCR but were negative by real-time RT–PCR. Sequencing was conducted on 23 influenza (H1N1)pdm09 isolates that were recovered from positive samples. Eight of these were negative for A(H1)pdm09 by real-time RT–PCR. There were two different mismatches in the probe target sites of the HA gene sequences of all isolates (n = 23) with additional mismatches only at position 7 (template binding site) identified for all eight negative real-time RT–PCR isolates. The prime target sites had no mismatches. Phylogenetic analysis of the HA gene showed that both the positive and negative real-time RT–PCR isolates were grouped in clade 6B.1; however, the real-time RT–PCR negative viruses were located in a subgroup that referred to substitution I295V.@*Conclusion@#Constant monitoring of genetic changes in the circulating influenza A(H1N1)pdm09 viruses is important for maintaining the sensitivity of molecular detection assays.

Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction primers

The purpose of this study was to develop Peptoniphilus mikwangii

Diseño y evaluación de primers in silico del gen E1 del virus de chikungunya para real-time PCR (qPCR) / Design and evaluation of primers in silico of the E1 gene of chukungunya virus for real -time PCR (q PCR)

El diseño de primers es una parte sustancial dentro de los ensayos de PCR, el mismo es aplicado en la caracterización de microorganismos, secuenciación, cuantificación, etc. En los últimos años se ha incrementado herramientas para este fin, se realizaron nuevas variantes, crearon nuevos enfoques, muchas de estas herramientas son de licencia comercial y otros de acceso libre y código abierto permiten al usuario mejorar o modificar según requerimiento el diseño de primers. En este trabajo comparamos las características básicas dentro del diseño de primers, empleando 2 tipos de herramientas web de acceso libre y código abierto: Primer-BLAST y Primer3Plus, se realizó el diseño in silico, análisis de estructuras secundarias y verificación de la especificidad de los primers obtenidos. Los resultados mostraron que el uso de variables adicionales o uso de penaltis mediante el software Primer3Plus afectan en la especificidad, formación de estructuras secundarias de los primers y de los amplicones. Además, se determinó que los parámetros que tienen muchos software por defecto, no aseveran el diseño de primers convenientes y/o específicos, en este sentido es importante realizar el uso de penaltis. Asimismo, se identificó que el uso de Primer3Plus ha permitido disminuir la formación de estructuras secundarias, sin afectar la especificidad de amplificación del marcador seleccionado.

Primer design is a substantial part of PCR assays, which is used in characterization of microorganisms, sequencing, quantification, etc. In recent years, new tools have been added for this purpose, new variants and approaches have been created, many of these tools are commercial and others are free including open source, giving the possibility to improve or modify as required. In this work, we compared the basic characteristics of the primers design, using 2 types of free and open source web tools, PrimerBLAST and Primer3Plus, in this sense the in silico design, analysis of secondary structures and verification of primers designed. The results showed that the use of additional variables or use of penalties using the software Primer3Plus affect in specificity, secondary structures formation in primers and in the products of amplification. In addition, default parameters, that many software have, do not imply that suitable and / or specific primers can be obtained, meaning the importance of the use of penalties. Therefore, the use of Primer3Plus has allowed to decrease the formation of secondary structures without affecting the amplification specificity of the selected marker.

Cross-amplification of heterologous microsatellite markers in Piracanjuba / Amplificação cruzada de marcadores microssatélites heterólogos em piracanjuba

ABSTRACT: Brycon orbignyanus, popularly known in Brazil as piracanjuba, is a fish with great economic value but whose natural population drastically decreased in number during the last years. In this context, genetic variability studies of natural stocks and in restocking programs are fundamental for the adoption of conservation measures. Current analysis verifies the cross-amplification of heterologous primers in B. orbignyanus. Fifty-two primers of the species Brycon opalinus, Brycon hilarii, Brycon insignis, Prochilodus sp., Piaractus mesopotamicus, Colossoma macropomum and Oreochromis niloticus were tested. Primers with the best reproducibility were applied to a sample of 20 individuals and the genetic parameters were calculated. Nine primers provided good results for cross-amplification with B. orbignyanus, involving (BoM5 and BoM13) of Brycon opalinus, (Bh5, Bh6, Bh8, Bh13 and Bh16) of Brycon hilarii, (Bc48-10) of Brycon insignis and (Par80) of Prochilodus argenteus. Primers of Piaractus mesopotamicus, Colossoma macropomum and Oreochromis niloticus failed to provide amplification or provided non-specificity. Results demonstrated the possibility of using primers of different species and genera of B. orbignyanus, facilitating genetic studies on the species.

RESUMO: A piracanjuba (Brycon orbignyanus) é um peixe de grande valor econômico que nos últimos anos tem apresentado uma redução drástica em suas populações naturais. Nesse contexto, estudos de variabilidade genética dos estoques naturais e nos programas de repovoamento são fundamentais para adoção de medidas conservacionistas. O objetivo do presente trabalho foi verificar a amplificação cruzada de primers heterologos em B. orbignyanus. Foram avaliados um total de 52 primers das espécies Brycon opalinus, Brycon hilarii, Brycon insignis, Prochilodus sp., Piaractus mesopotamicus, Colossoma macropomum e Oreochromis niloticus. Os primers com melhor reprodutibilidade foram aplicados a uma amostra de 20 indivíduos e os parâmetros genéticos foram calculados. Nove primers apresentaram resultados satisfatórios de amplificação cruzada com B. orbignyanus, sendo das espécies Brycon opalinus (BoM5 e BoM13), Brycon hilarii (Bh5, Bh6, Bh8, Bh13 e Bh16), Brycon insignis (Bc48-10) e Prochilodus argenteus (Par80). Os primers de Piaractus mesopotamicus, Colossoma macropomum e Oreochromis niloticus não apresentaram amplificação ou apresentaram inespecificidade. Os resultados revelaram a possibilidade da utilização de primers de diferentes espécies e gênero em B. orbignyanus, o que facilita a realização de estudos genéticos nessa espécie.

Estimation of genetic diversity and relationship in sugar beet pollinators based on SSR markers

Background: Genetic diversity studies are important for the selection of parents with a greater combination capacity that, when crossed, increase the chances of obtaining superior genotypes. Thus, 26 polymorphic simple sequence repeat (SSR) primers were used to assess the genetic diversity of 140 individual samples from 12 diploid sugar beet pollinators (pollen parents) and two cytoplasmic male sterile (cms) lines (seed parents). Eight pollinators originated from three research centers in the United States Department of Agriculture, while four pollinators and cms lines were from the Institute of Field and Vegetable Crops, Novi Sad, Serbia. Results: In total, 129 alleles were obtained, with a mean of 3.2 alleles per SSR marker. The observed heterozygosity ranged from 0.00 to 0.87 (mean = 0.30). Expected heterozygosity and Shannon's information index were the lowest for marker BQ590934 and the highest for markers SB15s and FDSB502s; the same markers were the most informative, with PIC values of 0.70 and 0.69, respectively. Three private alleles were found in pollinator EL0204; two in pollinator C51; and one in pollinators NS1, FC221, and C93035. Molecular variance showed that 77.34% of the total genetic variation was attributed to intrapopulation variability. Cluster and correspondence analysis grouped sugar beet pollinators according to the breeding centers, with few exceptions, which indicate that certain amount of germplasm was shared, although centers had their own breeding programs. Conclusions: The results indicate that this approach can improve the selection of pollinators as suitable parental components and could further be applied in sugar beet breeding programs.