Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

Antibodies to mycobacterial antigens for diagnosis of tuberculosis

In spite of the advent of several newer modalities in molecular and immunological methods of diagnosis, the diagnosis of smear negative tuberculosis is a major problem even today. Use of antibody detection ELISA for the purpose has been evaluated by many authors using a variety of antigens derived from mycobacteria. These antigens are reported to have many advantages and disadvantages. Here we review commonly used antigens used for detection of antimycobacterial antibodies for diagnosis of tuberculosis. Each antigen is reviewed for its application in diagnosis, sensitivity, specificity, cross-reactivity and clinical application.     

A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe array

AIMS: To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. METHODS AND RESULTS: A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC-6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88.5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR-ahpC intergenic region (86.5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88.5% and 100% for rifampicin resistance, and 86.5% and 100% for isoniazid resistance, respectively. CONCLUSION: A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. SIGNIFICANCE AND IMPACT OF THE STUDY: This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated.     

Natural Hidden Autoantibodies React with Negatively Charged Carbohydrates and Xenoantigen Bdi

Immunoglobulin preparations from sera of healthy donors contain polyspecific autoantibodies interacting with DNA and other charged antigens. These antibodies belong to the IgG class and can exist in the free or hidden state. The hidden antibody activity can be revealed after ion-exchange chromatography on QAE-Sephadex A-50. Immunoenzyme assay was used to assess the interactions of both free and hidden antibodies with different carbohydrates. The hidden antibodies were only able to interact with different polyanionic carbohydrates and neutral xenoantigen Bdi.     

基于多层网络控制的个体化癌症驱动基因识别方法

目的 识别癌症驱动基因,特别是罕见或个体特异性癌症驱动基因,对精准肿瘤学至关重要。考虑到肿瘤间的高度异质性,最近有一些方法尝试在个体水平上识别癌症驱动基因。然而,这些方法大多是将多组学数据整合到单一生物分子网络（如基因调控网络或蛋白质相互作用网络）中来识别癌症驱动基因,容易忽略不同网络所特有的重要相互作用信息。为了整合不同生物分子网络的相互作用数据,促进癌症驱动基因识别,迫切需要发展一种多层网络方法。方法 本文提出了一种多层网络控制方法 （PDGMN）,利用多层网络识别个体化癌症驱动基因。首先,利用基因表达数据构建针对个体病人的个体化多层网络,其中包括蛋白质相互作用层和基因相互关联层。然后,整合突变数据,对个体化多层网络中的节点进行加权。最后,设计了一种加权最小顶点覆盖集识别算法,找到个体化多层网络中的最优驱动节点集,以提高个体化癌症驱动基因的识别效果。结果 在三个TCGA癌症数据集上的实验结果表明,PDGMN在个体化驱动基因识别方面优于其他现有方法,并能有效识别个体病人的罕见癌症驱动基因。特别是,在不同生物分子网络上的实验结果表明,PDGMN能够捕获不同生物分子网络的独有特征,从而改进癌症驱动基因的识别结果。结论 PDGMN能有效识别个体化癌症驱动基因,并从多层网络的视角,加深我们对癌症驱动基因识别的理解。本文所用的源代码和数据集可以从https://github.com/NWPU-903PR/PDGMN获取。     

Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galα1-4GalNAcβ1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galα1-4GalNAcβ1-3Gal (NOR-tri), and weakly by Galα1-4Gal. However, the type 1 antibodies are strongly inhibited by Galα1-4Galβ1-3Gal-R and weakly by Galα1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galα1-4GalNAcβ1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several α-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-αGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.     

食源性致病菌多重分子生物学检测技术研究进展

快速、可靠的食源性致病菌高通量检测方法对于确保食品安全具有重要意义,近年基于DNA水平的多重分子生物学检测技术迅速发展,针对各种不同的食源性致病菌建立了多种多重分子检测技术,包括多重PCR、多重实时荧光PCR以及基因芯片等。对这些多重分子检测技术的最新研究进展作一综述,并且建议在今后该技术的研究中,仍需要在食品中多种致病菌同时选择性增菌培养、亚致死损伤修复以及检测内标的构建等方面取得突破,从而能够更好地实现食源性致病菌的高通量检测。     

DIRECT DETECTION OF SINGLE-NUCLEOTIDE POLYMORPHISMS IN BACTERIAL DNA BY SNPtrap

A major challenge with single-nucleotide polymorphism (SNP) fingerprinting of bacteria and higher organisms is the combination of genome-wide screenings with the potential of multiplexing and accurate SNP detection. Single-nucleotide extension by the minisequencing principle represents a technology that both is highly accurate and enables multiplexing. A current bottleneck for direct genome analyses by minisequencing, however, is the sensitivity, since minisequencing relies on linear signal amplification. Here, we present SNPtrap, which is a novel approach that combines the specificity and possibility of multiplexing by minisequencing with the sensitivity obtained by logarithmic signal amplification by polymerase chain reaction (PCR). We show a SNPtrap proof of principle in a model system for two polymorphic SNP sites in the Salmonella tetrathionate reductase gene (ttrC).     

Extensively drug-resistant tuberculosis: current challenges and threats

Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis caused by a Mycobacterium tuberculosis strain that is resistant to at least rifampicin and isoniazid among the first-line antitubercular drugs (multidrug-resistant tuberculosis; MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second-line drugs, namely amikacin, kanamycin and/or capreomycin. Recent studies have described XDR-TB strains from all continents. Worldwide prevalence of XDR-TB is estimated to be c. 6.6% in all the studied countries among multidrug-resistant M. tuberculosis strains. The emergence of XDR-TB strains is a reflection of poor tuberculosis management, and controlling its emergence constitutes an urgent global health reality and a challenge to tuberculosis control activities in all parts of the world, especially in developing countries and those lacking resources and as well as in countries with increasing prevalence of HIV/AIDS.     

7th Annual European Antibody Congress 2011

The 7th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The 2011 version of the EAC was attended by nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The first day focused on advances in understanding structure-function relationships, choosing the best format, glycoengineering biobetter antibodies, improving the efficacy and drugability of mAbs and epitope mapping. On the second day, the discovery of novel targets for mAb therapy, clinical pipeline updates, use of antibody combinations to address resistance, generation and identification of mAbs against new targets and biosimilar mAb development were discussed. Antibody-drug conjugates, domain antibodies and new scaffolds and bispecific antibodies were the topics of the third day. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.     

猪微卫星标记多重PCR扩增组合

采用多重PCR的方法,以快速扩增微卫星标记和节约试剂为目标,对其反应条件进行优化后,获得了46个扩增效果理想的微卫星标记多重PCR组合,其中30个为二重PCR,16个为三重PCR。实验结果表明,这些多重PCP反应的引物浓度为0.06~0.3μmol/L, Mg2+的浓度变化范围为1.5~3.0mmol/L,采用的PCR缓冲液的倍数为1.0、1.2、1.4或1.6,每个PCR反应聚合酶的用量在0.2和0.4U之间,退火温度及反应循环数分别为52~60℃ 和32~50℃。所有多重PCR进一步合并为17个可在ABI 337测序仪上进行电泳的组合。Abstract:In order to rapidly amplify pig microsatellite markers and save materials,multiplex PCR was used and its reaction condition was optimized.Forty-six combinations of multiplex PCR with good effects were obtained.Thirty of them are duplex-PCRs,sixteen are triplex-PCRs.The results of multiplexes showed that the concentration of primers varied among 0.06~0.3μmol/L,the Mg2+ concentration among 1.5~3.0 mmol/L;0.2~0.4 U of Taq polymerase and 1.0-,1.2-,1.4-,1.6-fold buffer were used,the annealing temperature and the cycle number varied among 52~60℃ and 32~50℃,respectively.All multiplexes were further combined into 17 sets for the electrophoresis on ABI 377 sequencer.     

结核分枝杆菌抗原检测研究进展

结核病仍然是一个严重的全球性公共卫生问题,有效控制结核病的障碍在于缺乏早期、准确的诊断方法。机体受到结核分枝杆菌感染后,体内首先出现的是结核分枝杆菌特异性抗原。因此,结核分枝杆菌抗原检测作为结核病早期诊断的方法可能具有很高的诊断价值。我们简要综述了结核分枝杆菌抗原检测的相关研究进展。