Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections.     

应用多重PCR方法检测石蜡包埋组织标本中的分枝杆菌DNA

应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感…     

Direct application of AvaII PCR restriction fragment length polymorphism analysis (AvaII PRA) targeting 644 bp heat shock protein 65 (hsp65) gene to sputum samples

To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS 6110 PCR. Although this method showed a sensitivity slightly lower than IS 6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS 6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.     

A new method of determining the virulence of nontubercular mycobacteria and weakly virulent Mycobacterium tuberculosis

A new method for enhancing the sensitivity of white mice to infection with faintly virulent mycobacteria is proposed. The method consists in the sensitization of animals with pertussis monovaccine introduced in a single-intraperitoneal injection 7 days before the infection of animals with the culture under study. The method permitted the detection of residual virulence in all opportunistic mycobacteria under study.     

High-sensitivity hybridization assay for quantitation of residual E. coli DNA

Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.     

Molecular inversion probes for sensitive detection of Mycobacterium tuberculosis

Nucleic acid-based detection of Mycobacterium tuberculosis infections has the potential to improve the analysis of the tuberculosis epidemiology and patient care by increasing the specificity and sensitivity of diagnosis. One potential diagnostic sequence, the DR locus, is present in all isolates of M. tuberculosis complex bacteria. It encodes no known gene product but is useful for molecular typing of M. tuberculosis because of its fortuitous absence in non-tuberculosis strains of mycobacteria. The DR locus contains a variable number of short direct repeats interspersed with non-repetitive spacers and is commonly used as a target for the spoligotyping method, a technique based on the detection of the presence or absence of distinct spacers between the repeats. In this study, we attempted to combine the specificity of molecular inversion probe (MIP) technology with the sensitivity of modified pyrosequencing readout in order to detect a short conserved 18 bp sequence included in DR locus in 25 isolates of M. tuberculosis. Additional sensitivity was obtained by introducing modifications in pyrosequencing methodology, by these means we achieved to detect 500 fg of M. tuberculosis DNA.     

An extremely rapid and simple DNA-release method for detection of M. tuberculosis from clinical specimens

A single-step, 5-min lysis method was investigated as a rapid technique to extract genomic DNA from mycobacteria for PCR detection of M. tuberculosis directly from clinical specimens. Of 67 smear-positive clinical specimens, 64 (95.5%) were positive by PCR after this rapid extraction method.     

Combining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000

TaqMan-nuclease assays are widely used for the qualitative detection of single nucleotide polymorphisms (SNPs) and the determination of biallelic states in pooled or heterozygous DNA samples. These assays are highly specific, reproducible, and suitable for high-throughput approaches. A crucial limitation of this method, and others, is the detection qf minor allele frequencies with detection limits of generally 3% to 9% for minor allele contributions. Here we describe the combination of customized TaqMan-nuclease assay and allele-specific restriction to increase the sensitivity of this method, allowing the qualitative detection of allele contributions as low as 0.05%.     

Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.     

Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples

AIMS: To develop a rapid, cost-effective and selective Alexandrium DNA extraction procedure from environmental samples in order to provide good-quality template for the downstream PCR-based detection assay. METHODS AND RESULTS: In this study, we tested a DNA extraction method based on silica-coated, superparamagnetic nanoparticles conjugated to a DNA-capture sequence (probe) complementary to a specific region of 5.8S rDNA of the genus Alexandrium. Cultured Alexandrium catenella cells were used as the harmful algal bloom species for the DNA extraction. Then, a PCR assay was performed with primers specific for the genus Alexandrium to assess the specificity and sensitivity of the nucleic acid extraction method. This method was applied to both cultured and field samples, reaching in both cases a detection limit of one A. catenella cell. CONCLUSIONS: The results suggest that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in cultured or environmental samples, ensuring sensitivity and specificity of the subsequent PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction method optimized in this study represents a progress towards the rapid and efficient direct detection of Alexandrium cells in seawater monitoring. In fact, this method requires no other equipment than a magnet and a hybridization oven and, in principle, can be adapted to different toxic microalgal species and can be automated, allowing the processing of a high number of samples.     

Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.     

Gene isolation with human T lymphocyte probes. Isolation of a gene that expresses an epitope recognized by T cells specific for Mycobacterium bovis BCG and pathogenic mycobacteria

We have used human CD4+ T lymphocyte clones as primary probes to identify and isolate lambda gt11 rDNA clones that express epitopes recognized by T cells. The method that we describe here permits a direct survey of T cell epitope coding sequences in genomic DNA or cDNA libraries. A lambda gt11 library of Mycobacterium leprae DNA was screened with M. leprae-reactive human T cell clones as probes, allowing the isolation of a M. leprae DNA clone encoding the unidentified Ag. This DNA clone differs in restriction maps from those previously identified by antibody probes and encodes an epitope that is unique to vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin and pathogenic mycobacteria. This method is generally applicable and should expedite the study of Ag and epitopes important to the T cell response in infections and in autoimmune diseases.     

Chromatographic determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA: a validation study

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable. The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.     

利用无传染性耻垢分枝杆菌建立小鼠结核病模型的探索

目的:利用耻垢分枝杆菌(M.smegmatismc2155)建立C57BL/6小鼠结核病模型。方法:每天以高剂量(5×107CFU)耻垢分枝杆菌给C57BL/6小鼠腹腔注射,连续感染4周,检测耻垢分枝杆菌对小鼠的致病性。分别于2周和4周处死小鼠,无菌条件下解剖小鼠取肺、脾脏组织匀浆,进行组织内细菌活力检测;通过嗜酸性染色进行分枝杆菌的鉴定;同时进行病理切片的制备,观察肺和脾脏组织的病理变化;最后进行菌体DNA的提取和基因检测,根据上述指标确定小鼠结核病模型的建立是否成功。结果:腹腔感染小鼠2周后,模型组小鼠只有脾脏组织匀浆液出现抗酸染色阳性菌落,肺部组织未见阳性菌落。腹腔感染小鼠4周后,模型组小鼠肺、脾脏组织匀浆液中均可见大量抗酸染色阳性的菌落;组织病理学观察结果显示:小鼠肺组织主要表现为以中性粒细胞为主的炎性病变;基因检测结果表明:模型组小鼠肺组织匀浆液中可检测到耻垢分枝杆菌特异性3-磷酸甘油醛脱氢酶(gap)基因,而脾脏组织未扩增出耻垢分枝杆菌特异性基因。结论:通过腹腔注射无致病性耻垢分枝杆菌方法,成功建立C57BL/6小鼠结核病发生模型,为结核分枝杆菌与宿主相互作用研究提供安全的疾病模型。     

Development of a novel,rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids

We describe the development of a molecular detection system designed for use with synovial fluid (SF)-based infections. The methodology employs a lysis/extraction procedure that effectively disrupts microorganisms allowing for release of the microbial DNA and its amplification by polymerase chain reaction (PCR). We tested the effectiveness of adding a mixed-bed, ion-exchange resin to the extract to remove PCR inhibitory components present in the SF. After centrifugation to separate the resin, DNA contained in the supernatant is subjected to PCR using oligonucleotide primers designed for broad-spectrum microorganism detection. Amplification products are analyzed by agarose gel electrophoresis and/or DNA hybridization methodology. We report here the detection sensitivity and specificity of the protocol using SF inoculated withEscherichia coli andStaphyloccocus aureus. We have applied this new methodology to clinical SF specimens with results superior to standard laboratory culturing assays.     

Chromatographic determination of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA: A validation study

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable.

The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.     

Evaluation of a low-cost calorimetric approach for rapid detection of tuberculosis and other mycobacteria in culture

Aims: The objective of this study was to evaluate the effectiveness of microcalorimetry in rapid detection of mycobacterium species using an inexpensive Isothermal microcalorimetry (IMC) instrument. In addition, we compared microcalorimetry with conventional monitoring techniques. Methods and Results: Isothermal microcalorimetry measures heat production rate and can provide rapid detection of living mycobacteria in clinical specimens. Using liquid medium showed that bacterial activity measured by IMC using a TAM Air^® agreed with the triphenyl tetrazolium chloride (TTC) assay. Using solid medium to enhance growth, fast‐growing mycobacteria detection was achieved between 26 and 53 h and slow‐growing mycobacteria detection was achieved between 54 and 298 h. In addition, the calorimetric data were analysed to estimate the growth rate and generation time of the mycobacteria monitored. Significance and Impact of the Study: Infections caused by mycobacteria are severe and difficult to treat. With 9·27 million new cases of tuberculosis in 2007, developing countries experience severe health and economic consequences owing to the lack of an affordable, fast detection method. Research‐grade IMC instruments are too expensive to use in developing countries. Our study demonstrates that less‐expensive instruments such as the TAM air ^® are adequate for mycobacteria detection and therefore establishes a clear proof of concept.