Detecção de Aeromonas spp. e do gene de virulência aerolisina em tilápias do Nilo (Oreochromis niloticus) com a técnica de mPCR / Detection of Aeromonas spp. and virulence gene aerolysin in Nile tilapia (Oreochromis niloticus) using PCR technique

As infecções causadas por bactérias do gênero Aeromonas estão entre as doenças mais comuns em peixes cultivados em todo o mundo, com ocorrência de aeromoniose em todos os países que possuem cultivo de tilápia do Nilo (Oreochromis niloticus). O presente trabalho descreve o desenvolvimento de uma nova multiplex PCR (mPCR) para diagnóstico de Aeromonas spp. e identificação do gene aerolisina (aerA). Para padronização da mPCR foram utilizadas cepas de referência de várias espécies do gênero Aeromonas e de outros gêneros. Também foram usadas cepas de campo de A. hydrophila oriundas de cultivos de peixes pacamãs (Lophiosilurus alexandri) e Aeromonas spp. de tilápias do Nilo. Os primers foram desenhados com base na região 16S rRNA e aerA. Para verificar a melhor temperatura de anelamento foram utilizados gradientes entre 59°C a 61°C com 40ng de DNA molde. Os produtos da amplificação da região 16S rRNA e do gene aerA apresentaram 786 e 550pb, respectivamente. A mPCR apresentou melhor temperatura de anelamento a 57,6°C com limite de detecção das concentrações de DNA em ambos genes (16S rRNA and aerA) de 10-10g/μL. A mPCR padronizada é rápida, sensível e específica no diagnóstico de Aeromonas spp. e identificação do gene aerolisina. Esta metodologia apresenta vantagens quando comparada aos métodos de diagnóstico convencionais, podendo ser utilizada em cultivos comerciais de tilápias do Nilo ou outros peixes. A identificação do gene aerolisina é uma importante ferramenta na determinação do potencial patogênico dos isolados de Aeromonas spp. estudados.(AU)

Infections caused by bacteria of the genus Aeromonas are among the most common diseases in fish farming systems worldwide, and this disease occurs in all countries which have Nile tilapia (Oreochromis niloticus) farmed. The present work describes the development of a new multiplex PCR (mPCR) technique that diagnosis the genus Aeromonas and detects aerolysin gene (aerA). Reference strains of several Aeromonas species and other genera were used for standardization of mPCR. Strains of A. hydrophila from "pacaman" fish (Lophiosilurus alexandri) and Aeromonas spp. from Nile tilapia from farming systems were used too. Primers were designed based on the 16S rRNA region and aerA (aerolysin toxin). To verify a better annealing temperature were used gradients between 59°C and 61°C with 40ng of the DNA template. The 16S rRNA gene and the aerA gene amplification products showed 786 and 550 bp, respectively. The mPCR showed better annealing temperature at 57.6°C, and the detection limit for both genes (16S rRNA and aerA) was 10-10g/μL of the DNA. The standardized mPCR is quick, sensitive, and specific for Aeromonas spp. diagnosis and to detect aerolysin gene. This method showed advantages when compared to the conventional diagnostic methods and can be used in Nile tilapia or other fish farming systems. The detection of aerolysin gene is an important tool to determine the potential pathogenicity of Aeromonas spp. isolates.(AU)

Molecular and Bacteriological Diagnosis of Mycoplasma Species Infection in Camels at Taif Governorate, Saudi Arabia

Aims: We designed this work to confirm if the PCR technique is more rapid and specific than traditional diagnostic method by culture. Study Design: In vitro experimental and molecular study. Place and Duration of Study: Genetic engineering and biotechnology unit, Taif University, Saudi Arabia from October, 2016 to September, 2017. Methodology: Ninety three nasal and tracheal swabs and lung samples were collected from camel in Taif slaughterhouse, Saudi Arabia. All samples were tested by culture and PCR method using universal primer of 16S rRNA gene. Results: There was no positive result obtained by culture method, but 30 (32.2%) of nasal swabs were positive using PCR method. Moreover, we used species-specific primers for Mycoplasma arginine, M. bovis and M. mycoides subspecies mycoides to identify the isolates at species level, but no positive results obtained with specific primers. These positive samples could be other Mycoplasma species. Conclusion: These results indicate that PCR technique is a specific molecular detection technique for Mycoplasma identification, and more sensitive test. These techniques are simple and fast methods to detect and isolate infected animals.

Effect of Guanxin-Shutong capsule on AT1、ERK2 expression in rats with chronic heart failure / 国际中医中药杂志

Objective By investigating the effects of AT1 and ERK2 signaling pathway in CHF,to study the effect of Guanxin-Shutong capsule on AT1,ERK2 expression in rats with chronic heart failure (CHF).Methods The CHF rat models were setup by coronary artery ligation,exhausting swimming and reducing feeding.Divided CHF rat methods into a model group,a lisinopril group,Qi-benefiting with Chinese medicine group,blood activating with Chinese medicine group,Qi benefiting and blood activating with Chinese medicine group(QBBA group) and Guanxin-Shutong capsule group.Rats without left coronary artery ligation were set for sham operated group.Real-time quantitative PCR technique and immunohistochemical method were adopted to detect AT1,ERK2 changes of CHF rats.Results AT1,ERK2 expression increased obviously in the model group compared with the sham operated group,and differences were statistically significant (P＜0.01).After treatment,AT1,ERK2 expression reduced significantly in QBBA group and Guanxin-Shutong capsule group than the lisinopril group.(P＞0.05).And therapeutic effect of Guanxin-Shutong capsule group was much better than other Chinese medicine treated group (P＜0.01).Conclusion Guanxin-Shutong capsule would achieve the goal of treating chronic heart failure By inhibiting the expression of AT1 and ERK2 in organizations,and inhibiting or reversing ventricular remodeling process.

Application PCR technique for analysis of fusion gene transcripts in the acute myelogenous leukemia

Background: In recent years, Vietnam has applied four methods (morphology, cell chemistry, immune marker classification, cyto genetic) in diagnosis and used multi-chemotherapy in treatment for acute myelogenous leukemia (AML)\r\n', u'Objectives: To initially determine some fusion gene transcripts in the acute myelogenous leukemia patients by applying PCR technique. Subject and method: The study included 19 patients with acute myelogenous leukemia treated in National Institute of Hematology and Blood Transfusion and Bachmai Hospital from April 2007 to August 2007. RNA were extracted from leukemic cells and PCR for AML1/ETO, CBFP/MYH11, PMR/RARa fusion transcript was done. Results: Number of male patients was 6 (32%), female patients was 13 (68%). The average age of these patients was 32.67 \xb113.62. There were three M4, M4eo patients with AML1/ETO gene (accounting for 16%), two M2, M4 patients with CBF/MYH1 gene and type F of genetic modification accounting for 11%), two M3 patients with PMR/RAR\u03b1 and Bcr3 of genetic modification (accounting for 11%). Conclusion: Results of the study did not differ significantly from other researches in the world. This study showed the need of applying the PCR technique in determining fusion gene transcript together with traditional cyto-genetic method.\r\n', u'\r\n', u'

The application of the PCR technique to find the DYZ gene for the Y chromosome determination

On 40 subjects of unspecified gender, PCR technique application for identifying DYZ1 gen to determine Y chromosome was performed in 38 subjects (95%). The results were conformed with PCR to identify TDF and the caryotype of the control group using PCR for detecting DYZ gen in determining Y chromosome. The accuracy reached 100% with no pseudonegative or pseudopositive results. The rest 5% (2 subjects) had not Y chromosome definitely. A case of TDF on X chromosome was detected affirmatively, as a first case in Vietnam.

Nested PCR definition of species composition of malaria parasites in Thanh commune, Huong Hoa district of Quang Tri province

Nested polymerase chain reaction (nested PCR) was applied to idennify species composition of four human malaria parasite species in Thanh commune, Huong Hoa district of Quang Tri province. The pair of primers specific to each species of P.falciparum, P.vivax, P.malariae and P.ovale were used. AND fragments specific to each species was 18ssr-RNA)[5], [10]. The analysis of 152 blood samples showed only two species of malaria parasites of P.falciparum (71%) and P.vivax (29%) present in the study area. Single P.falciparum infection rate was 65.8%, single P.vivax infection rate was 16.4% and the double infection of P.falciparum and P.vivax was 17.8%

The Study for 302 Cases of Chlamydia Genital Infection / 中国医师杂志

Objective To study whether the Chlamydia Trachomatis (CT) growth cycle affects the results of culture of CT infection, clinical symptom and the effectiveness of antibiotics .Methods The 302 cases from genecology out-patients were classified into two groups: the symptomatic ones (group A) and the asymptomatic another (group B), and The CT infection was examined by both PCR technique and culture medium, the positive cases were revisited twice later.Results ⑴There was no difference between two diagnostic methods in the group A. While in the group B, the positive rate of with PCR method was 8 91%(18/184), which was higher than that in culture medium 2 48%(5/197),P0 05. Total 96%(48/50) of cases from the double positive group revert to culture negative, only 36%(18/50) of cases were cured, while 60%(30/50) ones just get to the single positive group in PCR method.⑷After ceasing antibiotics for at least one month, those who have not been cured revisit again, 13 33%(4/30) of single positive cases with PCR method was reverted to double positive with PCR,culture medium methods.Conclusion CT infection in female genital system,positive determinable rate of PCR method is higher than that of culture medium method.

Genetype combination distribution and allele frequency distribution of single nucleotide polymorphisms of NOS2 C-1173T and G-954C in the Han Chinese population / 重庆医科大学学报

Objective:To study genetype combination distribution and allele frequency distribution of NOS2 C-1173T and G-954C single nucleotide polymorphisms (SNP) of inducible nitric oxide synthase(NOS2) in the Chinese Han nationality population.Methods:Genomic DNA of leukocytes in venous blood was obtained from 565 Han Chinese people(female 251 and male 314).The NOS2 G-954C and NOS2 C-1173T SNPs were genotyped by nested allele-specific primer (NASP)PCR.Results:The genotype frequencies of CC,CT and TT were 66.73%,26.37% and 6.90% respectively in the NOS2 C-1173T SNP.C and T allele haplotype frequencies of NOS2 C-1173T SNP was 79.88% and 20.12% respectively.The genotype frequencies of NOS2 GG,GC and CC were 61.77%,37.88% and 0.35% respectively in the NOS2 G-954C SNP.G and C allele haplotype frequencies was 80.71% and 19.29% respectively in the NOS2 G-954C SNP.The preceding 5 combinations in the natural combination distribution of two sites of NOS2 SNP were found as follows:(1)The genotype combination of 233 subjects was NOS2 C-1173C+G-954G,its probability being 41.24%.(2)The genotype combination of 143 subjects was NOS2 C-1173C+G-954C,its probability being 25.31%.(3)The genotype combination of 89 subjects was NOS2 C-1173T+G-954G,the probability being 15.75%.(4)The genotype combination of 59 subjects was NOS2 C-1173T+G-954C,the probability being 10.44%.(5)The genotype combination of 27 subjects was NOS2 T-1173T+G-954G,the probability being 4.78%.Conclusion:This study shows the features of combination distribution and frequency distribuion of NOS2 SNPs and provides the basic laboratory data for the further study relationships among NOS2 SNPs,its physiological function and diseases.