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).

DNA methylation diversity analysis of Lycium barbarum samples from different cultivation areas based on MSAP / 中国中药杂志

Obvious epigenetic differentiation occurred on Lycium barbarum in different cultivation areas in China. To investigate the difference and change rule of DNA methylation level and pattern of L. barbarum from different cultivation areas in China, the present study employed fluorescence-assisted methylation-sensitive amplified polymorphism(MSAP) to analyze the methylation level and polymorphism of 53 genomic DNA samples from Yinchuan Plain in Ningxia, Bayannur city in Inner Mongolia, Jingyuan county and Yumen city in Gansu, Delingha city in Qinghai, and Jinghe county in Xinjiang. The MSAP technical system suitable for the methylation analysis of L. barbarum genomic DNA was established and ten pairs of selective primers were selected. Among amplified 5'-CCGG-3' methylated sites, there were 35.85% full-methylated sites and 39.88% hemi-methylated sites, showing a high degree of epigenetic differentiation. Stoichiometric analysis showed that the ecological environment was the main factor affecting the epigenetic characteristics of L. barbarum, followed by cultivated varieties. Precipitation, air temperature, and soil pH were the main ecological factors affecting DNA methylation in different areas. This study provided a theoretical basis for the analysis of the epigenetic mechanism of L. barbarum to adapt to the diffe-rent ecological environments and research ideas for the introduction, cultivation, and germplasm traceability of L. barbarum.

Identification of Andrographis Herba and its common products using mini-barcode / 中国天然药物

Andrographis Herba, the aerial part of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), has a wide geographic distribution and has been used for the treatment of fever, cold, inflammation, and other infectious diseases. In markets, sellers and buyers commonly inadvertently confuse with related species. In addition, most Chinese medicinal herbs are subjected to traditional processing procedures, such as steaming and boiling, before they are sold at dispensaries; therefore, it is very difficult to identify Andrographis Herba when it is processed into Chinese medicines. The identification of species and processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA barcoding has received considerable attention as a new potential means to identify species and processed medicinal materials. In this study, 17 standard reference materials of A. paniculata, 2 standard decoctions, 27 commercial products and two adulterants were collected. Based on the ITS2 sequence, it could successfully identify A. paniculata and adulterants. Moreover, a nucleotide signature consisting of 71 bp was designed, this sequence is highly conserved and specific within A. paniculata while divergent among other species. Then, we used these new primers to amplify the nucleotide signature region from processed materials. In conclusion, the DNA barcoding method developed in the present study for authenticating A. paniculata is rapid and cost-effective. It can be used in the future to guarantee the quality of Andrographis Herba of each regulatory link for clinical use.

Development of tetra-primer ARMS-PCR protocol to genotype the prolactin receptor SNP 39136666 and assessment of this SNP in Brazilian locally adapted cattle breeds / [Desenvolvimento de protocolo de tetra-primer ARMS- PCR para genotipagem do SNP 39136666 do receptor da prolactina e avaliação desse SNP em raças bovinas brasileiras localmente adaptadas]

As raças taurinas de origem ibérica Limonero e Carora (Bos primigenius taurus) possuem o fenótipo de pelo curto, liso e com baixa densidade folicular, o que confere a esses animais maior tolerância térmica e melhor produtividade em regiões quentes. Diferentes mutações associadas a esse fenótipo foram descritas no gene do receptor de prolactina PRLR, localizado no cromossomo bovino BTA20. Uma mutação recentemente encontrada é a substituição do nucleotídeo C por T, SNP 39136666 (p. R497*), no exon 11, que gera um códon de parada e, consequentemente, uma menor isoforma desse receptor. Neste trabalho, desenvolveu-se um protocolo rápido e de baixo custo para detecção desse SNP, utilizando-se a técnica de tetra-primer ARMS-PCR. Assim, foi possível detectar essa mutação nas raças brasileiras de origem ibérica localmente adaptadas: Caracu, Crioulo Lageano, Mocho Nacional e Pantaneiro. O alelo T foi mais frequente na raça Caracu (80%), enquanto o alelo C foi mais frequente na raça Crioulo Lageano (84%). Essa simples metodologia pode ser usada para genotipar esse SNP e ajudar na aplicação dessas informações moleculares em programas de melhoramento focados na tolerância térmica em bovinos taurinos e seus mestiços.(AU)

Development of nuclear SSR and chloroplast genome markers in diverse Liriodendron chinense germplasm based on low-coverage whole genome sequencing

BACKGROUND: Liriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm. RESULTS: We performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79. CONCLUSIONS: The genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Optimized inverse PCR strategy for constructing multilocus mutants efficiently / 生物工程学报

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.

Comparison of intestinal microbial community succession based on different universal primer sets / 生物工程学报

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.

Rapid, specific, and sensitive detection of the ureR_1 gene in Klebsiella pneumoniae by loop-mediated isothermal amplification method

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.

Mutation Spectrum of FANCJ Gene in Adult Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To detect and analyze the mutation status of FANCJ gene in adult AML patients, so as to provide the basis for studying the mechanism of FANCJ driven AML and guiding the preventim and treatment of deseese.@*METHODS@#The cDNAs were extracted and transeripted from bone marrow cells and normal skin cells in 222 newly diagnosed AML patients. The primers were designed for FANCJ gene coding region, the mutations of FANCJ gene coding region in AML patients as well as the mutations of FANCJ gene in mucous membrane epethelia in patients were detected by PCR and sanger seguencing; the evolutionary conservation of FANCJ mutation in different organisms was analyzed by NCBI Blast online bioinformaties software.@*RESULTS@#The sequencing analysis showed that the mutations of FANCJ gene happened in 11 sites of FANCJ gene coding region, which were as followed: exon5:c.G430A:p.A144T, exon6:c.A587G:pN196S, exon9:c.C1255T:p.R419W, exon10:c.G1442A:p.G481D, exon11:c.C1609G:p.L537V, exon16:c.C2360T:p.P787L, exon17:c.C2440T:p.R814C, exon19:c.C2608T:pH870Y, exon19:c.A2686G:p.I896V, exon19:c.C2830G:p.Q944E, exon20:c.G3412A:p.D1138N. Among them, the repeatability existed in mutations of A144T, N196S, R814C, I896V and Q944E. Beside, the mutation sites of A144, R419, G381, L537, P787, H870, Q944 and D1138 were highly conserved in different organisms.@*CONCLUSION@#Among 222 adult AML patients, the mutations of FANCJ gene have been found in 26 patients, moreover, the mutation sites are relatively conserved in different organisms, and possess important fanction. The results of this study provide the basis for exploring the mexhanism of FANCJ gene driven AML and for guiding the prevantion and treatment of AML.

Rapid identification of Cordyceps and its products using DNA barcoding method / 中国中药杂志

Cordyceps is one of the most valuable traditional Chinese medicines. There are various counterfeits in markets because of high price and limited output. In this study,116 Cordyceps,146 hosts and 29 related products were collected and detected by using normal DNA barcoding technology and specific PCR method. The results indicated that Cordyceps and its adulterants could be distinguished from each other through DNA barcoding technology based on ITS and COⅠsequences. Two pairs specific primers ITSSF1/ITSSR1 and ITSSF2/ITSSR2 were developed to amplify 297 bp and 136 bp ITS regions of Cordyceps sinensis,respectively. It could be used to identify C. sinensis specifically and rapidly. Furthermore,specific primers ITSSF1/ITSSR1 and ITSSF2/ITSSR2 combined with ITS and COⅠsequences could differentiate powder Cordyceps from fermentation mycelia and could identify related products. Therefore,the method developed from this study could be applied to identify the powder of Cordyceps from fermentation mycelia and related products efficiently.

Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP / 生物工程学报

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.

Establishment of a novel TB-ARMS qPCR method for kras mutation detection / 生物工程学报

A simple, robust and highly sensitive TB-ARMS method based on qPCR technique was developed to detect kras mutations. The technique was evaluated, and its clinical application was investigated. Mutation specific primers for eight common kras mutations and wild type gene targeted blockers were designed and optimized. Moreover, a mutant-enriched condition was used in to improve the sensitivity and specificity of mutation detection. Constructed plasmids carrying mutant kras genes, as well as confirmed wild type genomic DNA, were used as standard samples for evaluation of the methodology. The performance of our new method was validated by comparing the results of our method with that of a commercial kras kit in testing 40 clinical samples. Preoperative plasma samples, as well as paired tissue samples, were tested in parallel for evaluation of its clinical application. We have developed a new TB-ARMS method for kras mutation detection that can detect minor mutant alleles with a frequency as low as 0.01% in a heterogeneous sample. We have successfully demonstrated its 0.01% detection sensitivity with highly specific mutant amplification in conjunction with selective wild type suppression by blocker under a mutant-enriched reaction condition. We also showed that our TB-ARMS method was more accurate than the commercial kras kit, which is widely used presently. Furthermore, we have validated our method as an efficient liquid biopsy method, and the results of the plasma DNA detection with our TB-ARMS method were in consistent with the sequencing results of paired tissue samples. In conclusion, our TB-ARMS qPCR method could be effectively applied in kras mutation test for clinical tissue samples, as well as for liquid biopsy samples such as plasma.

Phylogeny of the Trichogramma endosymbiont Wolbachia, an alpha-proteobacteria (Rickettsiae) / Filogenia do endosimbionte Wolbachia em Trichogramma, an alpha-proteobacteria (Rickettsiae)

Abstract Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.

Resumo Endosimbiontes do gênero Wolbachia (Hertig) são extensivamente estudados em uma ampla gama de organismos e são conhecidos por serem transmitidos via citoplasma do ovo hospedeiro para seu descendente. Wolbachia pode causar vários tipos de alterações reprodutivas nos artrópodes. Nas espécies de Trichogramma, a reprodução partenogenética induzida por Wolbachia, possibilita as fêmeas dos parasitoides a produção de fêmeas a partir de ovos não fertilizados e estas bactérias estão presentes em pelo menos 9% de todas as espécies de Trichogramma. Estudos filogenéticos têm levado a subdivisão do clado Wolbachia em cinco supergrupos (A, B, C, D and E). Wolbachia em Trichogramma pertence ao supergrupo B. Com o gene wsp foi possível se distinguir quatro grupos de Wolbachia que infectam Trichogramma e adicionar um novo grupo (Ato) devido a inclusão de Wolbachia detectada em Trichogramma atopovirilia (Oatman and Platner, 1983). Primers específicos foram construídos e testados para o grupo "Ato". Setenta e cinco por cento de todas as linhagens de Wolbachia que infectam Trichogramma se enquadraram dentro do grupo "Sib".

Optimización de una técnica de PCR convencional para detección de virus de papiloma humano tipo 16 y 18 / Optimization of a conventional PCR technique for detection of human papillomavirus type 16 and 18

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11ng/µL para VPH 16 y 21,7x10-12ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano(AU

Cervical cancer is the second most common female cancer worldwide. It is caused by the human papilloma virus (HPV). Thirteen genotypes of high oncogenic risk human papilloma viruses (HPV-HR) have been identified, among which types 16 and 18 are the most frequently detected in cervical cancer. In Paraguay, they are detected in 70% of the invasive cancer cases. Therefore, the objective was to standardize and determine the detection limit of a conventional PCR technique for the detection of HPV 16 and 18. Better results were observed with 2mM MgCl2 and 60°C for the alignment temperature in detection of HPV 16 and 18 DNA. The limit of detection was 14.6x10-11ng/µL for HPV 16 and 21.7x10-12ng/µL for HPV 18. This work will help other studies for the detection and identification of these viral types by PCR in order to identify a group of HPV-HR positive women who have higher risk for the development of lesions and cervical cancer and need a closer follow-up(AU)

Diferenciación de serovares de leptospiras patógenas mediante PCR del gen ligB y secuenciación / Differentiation of pathogenic leptospires spp by PCR of ligB gene and sequencing

La leptospirosis es la zoonosis de mayor distribución mundial. El objetivo del preReacción en cadena sente trabajo fue desarrollar una técnica molecular que diferencie leptospiras patógenas. Se amplificó mediante PCR y se secuenció una región de la adhesina ligB, presente solo en las especies patógenas. Se utilizaron los iniciadores ligBFpet y ligBRpet. Dichos iniciadores lograron amplificar el ADN blanco de 6 cepas patógenas referenciales de Leptospira interrogans (serovar Pomona cepa Pomona, serovar Canicola cepa Hond Utrecht IV, serovar Copenhageni cepa M 20, serovar Wolffi cepa 3705, serovar Pyrogenes cepa Salinem y serovar Hardjo cepa Hardjoprajitmo) y el de Leptospira borgpetersenii serovar Castellonis cepa Castellon 3; también el de 4 cepas patógenas aisladas de bovino, porcino, rata y comadreja. En las 2 cepas referenciales no patógenas utilizadas, Leptospira biflexa serovar Patoc cepa Patoc i y L. biflexa serovar Andamana cepa Andamana, no hubo amplificación. La secuenciación de los productos amplificados expuso una suficiente variación entre los serovares estudiados, que permite diferenciarlos.

Leptospirosis is a zoonosis having worldwide distribution. The objective of this work was to develop a molecular technique to differentiate pathogenic Leptospira spp. A region of adhesin ligB, present only in the pathogenic species was amplified by PCR and sequenced. ligBRpet and ligBFpet primers were used, which amplified the target DNA from pathogenic L. interrogans reference strains serovars Pomona strain Pomona, Canicola strain Hond Utrecht IV, Copenhageni strain M 20, Wolffi strain 3705, Pyrogenes strain Salinem, Hardjo strain Hardjoprajitmo, L. borgpetersenii serovar Castellonis strain Castellon 3 and 4 pathogenic strains isolated from bovines, pigs, rats and opossums. L. biflexa serovars Patoc strain Patoc i and Andamana strain Andamana were not amplified. Sequencing of the amplified products exhibited sufficient variation among serovars, which differentiates them.

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

Estandarización de una prueba múltiple de reacción en cadena de la polimerasa en tiempo real para la identificación de Angiostrongylus cantonensis, A. costaricensis y A. vasorum / Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Resumen Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específica para el manejo de las angiostrongilosis humana y la animal. Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus. Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®. Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano. Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %). Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

Abstract Introduction: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. Objective: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. Materials and methods: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. Results: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). Conclusions: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.