Accomplishment of a protocol for simultaneous detection of 14 intestinal bacterial pathogens based on PCR-Reverse Dot Blot

Food poisoning, caused by a bacterial infection, consequently led to a wide range of infections and endanger to public health, has been considered as a big concern in the world. Therefore, it is an urgent demand for the clinical laboratory to exactly identify infectious bacteria. In our previous study, we successfully published a protocol based on the PCR-Reverse Dot Blot (PCR-RDB) to determine simultaneously 12 bacterial intestinal pathogens, including Bacillus cereus, Clostridium botulinum, Clostridium perfringen, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157: H7, Salmonella spp., Shigella spp.., Vibrio cholerae, Vibrio parahaemolyticus, Yersinia enterocolitica and Brucella spp. In this study, we continuously developed our published protocol by designing additional probes: positive control probe, negative control probe, color control probe and background signal controller. Moreover, concerning the clinical demand, the supplement of two designed probes, which detected Campylobacter jejuni và Yersinia enterocolitica O:8 caused intestinal infected diseases mainly in children, was necessary. As a result, this completed PCR-RDB protocol can simultaneously detect a total of 14 intestinal bacterial pathogens within high specificity and the sensitivity of 102 copies/ml. For the protocol confirmation, it was tested by 30 fecal samples and the results completely match with the commercial kit PowerCheckTM 20 Pathogen Multiplex Real-time PCR Kit (Korea)

TÁC ĐỘNG CỦA MANNITOL ĐẾN KHẢ NĂNG SINH TRƯỞNG VÀ PHÁT TRIỂN CỦA CÂY NHA ĐAM (ALOE VERA L.) TRONG ĐIỀU KIỆN NUÔI CẤY IN VITRO

Bài báo này trình bày kết quả đạt được trong nghiên cứu ảnh hưởng của mannitol đến sự sinh trưởng và phát triển của cây nha đam (Aloe vera L.) nuôi cấy in vitro. Đoạn thân và đỉnh sinh trưởng tách từ cây nha đam in vitro được cấy lên môi trường cơ bản Murashige và Skoog (MS) có bổ sung 8 g/l agar, 30 g/l saccharose, 1g/l than hoạt tính, 1,0 mg/l BAP kết hợp với 0,5 mg/l NAA, pH 5,8. Trên môi trường này đoạn thân và đỉnh sinh trưởng của cây nha đam in vitro tái sinh cụm chồi rất tốt với trung bình tương ứng là 11,13 chồi/mẫu và 12,2 chồi/mẫu. Chồi đơn tách từ cụm chồi in vitro tạo rễ, sinh trưởng và phát triển tốt trên môi trường MS có bổ sung 0,5 mg/l NAA. Cây nha đam in vitro được xử lý với manitol ở các nồng độ thay đổi từ 1-16% để gây hạn sinh lý trong khoảng thời gian khác nhau. Kết quả cho thấy khi xử lý mannitol ở nồng độ thấp (1-4%) ít gây ảnh hưởng đến khả năng sinh trưởng và phát triển của mẫu nha đam in vitro, tuy nhiên khi xử lý mannitol ở nồng độ cao hơn (8, 10, 14%) gây giảm mạnh khả năng sinh trưởng và phát triển của mẫu nuôi cấy, đặc biệt khi xử lý mannitol ở nồng độ 16% gây tổn thương rất lớn đến hình thái lá và khả năng sinh trưởng của mẫu thí nghiệm. Từ khóa: in vitro, mannitol, nha đam, phát triển, sinh trưởng

Establisment of realtime pcr protocol based on 16s rrna gene of mycobacterium tuberculosis

Currently, tuberculosis is still one of the most common infectious diseases, mainly caused by Mycobacterium tuberculosis bacterium infection. PCR is a molecular biology technique used to detect infectious agents, especially viruses or bacteria with slowly culture as in the case of Mycobacterium tuberculosis because of its fastness, accurateness and high sensitivity. We carried out this study in order to develop a protocol based on Realtime PCR using 16S rRNA gene as a target sequence which is extensively used in taxonomy, molecular evolution and medical diagnostics. We have succeeded in designing the primers/probes on the 16S rRNA gene to detect Mycobacterium tuberculosis by Realtime PCR; have identified the parameters of the specificity of the primers/probe, theoretically and experimentally; the sensitivity of the primers/probe in the Realtime PCR protocol reached to 102 copies/ml; the protocol was also tested on 30 samples given good results

Establisment of Methodology for Identification of Insect-Parasitic Fungi by Molecular Analysis on ITS1-5.8S-ITS2 Region

Insect-parastitic fungi, especially Cordyceps sinensis, parasitize larvae of insects, are used as traditional medicinal drugs in various Asian countries, including Vietnam. However, knowledge of taxonomy of these fungi is still limited, due to many factors such as the difficulties in the classification and identification. Recently, the identification of these fungi was improved by molecular and bioinformatic methods. In this preliminarystudy, first we want to establishthe protocol including DNA extraction, PCR, sequencing then proof-reading and phylogenetic tree construction. The target sequence used for this protocol is Internal transcribed spacer –ITS. The protocol is carried out using the sample set, in which the already identified results by morphology and molecular biology combined bioinformatics were in high corcordance. Therefore, this protocol will be applied in order to identifyinsect-parasitic fungi specimens collected from Langbian mountain, Dalat, Lamdong, Vietnam

Application of real-time PCR to characterize the viral load, genotypes, resistance of hepatitis B virus at Dong Thap hospital

Hepatitis B is caused by the hepatitis B virus (HBV) and may be either acute or chronic. Chronic hepatitis B virus (HBV) infection is the major global cause of chronic hepatitis, as a result, which leads to cirrhosis and increases the risk of liver cancer development (hepatocellular carcinoma). Recent advances in the diagnosis and treatment of hepatitis B have enormously contributed to reduce the side effects of this disease in which the real-time PCR technique had the significant contribution todiagnosis, monitoring and therapy. In the current study, therefore, our purpose is the application of real-time PCR method to detect hepatitis B viral load, genotypes as well as lamivudine (LAM) and adefovir (ADV) resistance in patients at Dong Thap Hospital. The results figured that the mean age of HBV patients was 37.0 ± 0.34, the proportion of male infected was 59.0%. In addition, our results also showed that the rate of the patients with the normal ALT level and HBeAg negative were 51.2% and 23.0%, respectively. Moreover, the patients who were not indicated for treatment were approximately 30.0%. Among the group of HBsAg positive, the rate of HBV-DNA positive was 82.0% and a majority rate of 59.1% was HBV genotype B. The group of high viral load in those samples was equal to or greater than 20,000 IU/ml (36.0%). The total rate resistant mutation occurred in 56.1%, the rate of LAM resistant mutations was the most value of 86.0% and ADV resistant mutations were 68.0%. LAM resistant mutation 204I occurred at 84.0%. ADV resistant mutation A181T was the highest rate of 68.0%. Resistance mutations often associated with a higher proportion of HBeAg positive and the high viral loads. Meanwhile, the influence of genotype infections on the clinical, para-clinical characteristics were not clear