Laser-assisted microdissection for real-time PCR sample preparation

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.     

Laser-assisted microdissection of the kidney: Fundamentals and applications

Laser-assisted microdissection (LAM) permits the procurement of relatively pure cell populations from histological sections. When applied to the kidney, LAM combined with molecular biological techniques has expanded our understanding of renal biology and pathology. Both frozen and fixed renal tissues can be microdissected. However, sample type and tissue processing can influence the quality of molecular data generated. Data analysis may also be complicated by relative variations in gene expression levels. Importantly, preliminary studies have shown that molecular data obtained following LAM on the kidney can offer new diagnostic and prognostic information. Thus, LAM and molecular markers may eventually become incorporated into the routine kidney biopsy examination.     

Be more specific! Laser-assisted microdissection of plant cells

Laser-assisted microdissection (LAM) is a powerful tool for isolating specific tissues, cell types and even organelles from sectioned biological specimen in a manner conducive to the extraction of RNA, DNA or protein. LAM, which is an established technique in many areas of biology, has now been successfully adapted for use with plant tissues. Here, we provide an overview of the processes involved in conducting a successful LAM study in plants and review recent developments that have made this technique even more desirable. We also discuss how the technology might be exploited to answer some pertinent questions in plant biology.     

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.     

Two-tube multiplex real-time reverse transcription PCR to detect six human coronaviruses

正Dear Editor,Coronaviruses are enveloped positive-strand RNA viruses with 27–33 kb genomes.These viruses are classified into four genera,namely Alphacoronavirus,Betacoronavirus,Gammacoronavirus,and Deltacoronavirus     

SYBR green real-time PCR method to detect Clostridium botulinum type A

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).     

A real-time PCR to detect and analyze virulent EMCV loads in sows and piglets

A real-time polymerase chain reaction with SYBR Green was developed for the detection and quantification of encephalomyocarditis virus (EMCV) in porcine tissues; the method uses two primers specific for the 3D gene. The detection limit of this assay was 22 gene copies/reaction, equivalent to 0.001?TCID₅₀/ml. The assay was linear over a 10⁷ dilution range of template concentrations and was specific for EMCV; it did not amplify other porcine pathogens (porcine circovirus 2, porcine reproductive and respiratory virus, classical swine fever virus, pseudorabies virus, or porcine teschovirus). This assay detected EMCV titers at least 10⁴ smaller than the routine PCR assay. To increase our understand of EMCV pathogenesis, the new method was used to quantify levels of EMCV genome in various tissues of artificially challenged sows and piglets. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. The real-time PCR method described here should be useful for the study of EMCV infection and distribution in pigs.     

Use of real-time PCR to detect canine parvovirus in feces of free-ranging wolves

Using real-time PCR, we tested 15 wolf (Canis lupus) feces from the Superior National Forest (SNF), Minnesota, USA, and 191 from Yellowstone National Park (YNP), USA, collected during summer and 13 during winter for canine parvovirus (CPV)-2 DNA. We also tested 20 dog feces for CPV-2 DNA. The PCR assay was 100% sensitive and specific with a minimum detection threshold of 10(4) 50% tissue culture infective dose. Virus was detected in two winter specimens but none of the summer specimens. We suggest applying the technique more broadly especially with winter feces.     

基于TaqMan探针的蜜蜂囊状幼虫病病毒荧光PCR检测方法的建立和应用

为建立快速有效的蜜蜂囊状幼虫病检疫方法, 依据TaqMan荧光标记探针技术原理, 针对蜜蜂囊状幼虫病病毒保守序列, 设计出一对特异性引物和一条探针, 建立了一种快速检测蜜蜂囊状幼虫病病毒的荧光PCR方法。该方法对蜜蜂囊状幼虫病的检测具有较好的特异性, 与蜜蜂急性麻痹病病毒、蜜蜂慢性麻痹病病毒、蜜蜂残翼病病毒和黑蜂王台病病毒之间均无交叉反应。检测灵敏度可达1.0×10²拷贝/μL阳性质粒, 可对低病毒含量的样品进行准确检测。重复性和稳定性试验结果显示, 变异系数为1.6%, 说明该方法具有较好的重复性和稳定性。应用该方法对蜜蜂及蜂制品进行检测, 结果显示所建立的荧光PCR检测方法4 h内即可报告检测结果, 该方法具有快速、灵敏、特异及重复性好等优点, 适用于蜜蜂及其制品中蜜蜂囊状幼虫病病毒的快速检疫。     

Laser-assisted microdissection for the study of the ecology of parasites in their hosts

The population biology of internal parasites is difficult to study because the adult parasites are often inaccessible, deep within the host's body. Developing stages, such as eggs in the faeces or larvae in the skin are more easily obtained, but are difficult to handle because they are often very small and with a tough cuticle. This has limited their use in molecular ecology for estimating population biology parameters of the adults (their parents). We have used Onchocerca ochengi (a filarial nematode parasite of cattle) to describe a novel and generally applicable method of easily and conveniently isolating individual larvae (microfilariae) from the host using laser‐assisted microdissection. Furthermore, we have been able to improve the isolation of DNA by using the laser to bisect the larva to release DNA from the tissues enclosed within the parasite cuticle, and in this way we have achieved amplification of fragments over 1400 bp, and routinely PCR‐amplified single‐copy sequences from 5% of the DNA from a single larva (the equivalent of approximately 15 nuclei), and regularly from 0.5%.     

Indirect detection of Bacillus anthracis using real-time PCR to detect amplified gamma phage DNA

Typical real-time PCR methods used to identify Bacillus anthracis do not distinguish between viable and non-viable spores, which would be critical in any first response and remediation scenarios. This study combined both real-time PCR, using primers specifically designed for gamma phage, with the highly specific gamma phage amplification into one simple assay to indirectly detect Bacillus anthracis. Since the amplification of gamma phage only occurs in the presence of a suitable host, the detection of increasing concentrations of progeny gamma phage DNA using real-time PCR implies the presence of viable Bacillus anthracis cells. This method detected a starting Bacillus anthracis concentration of 207 cfu/mL, equivalent to less than one cell in 20 microL, in less than 5 h.     

Immunocytochemistry and laser capture microdissection for real-time quantitative PCR identify hindbrain neurons activated by interaction between leptin and cholecystokinin.

Current evidence suggests that leptin reduces food intake in part by enhancing the hindbrain neuronal response to meal-related gastrointestinal signals, including cholecystokinin (CCK), but the phenotypes of the relevant cells are not known. To identify neurons that participate in this interaction in the rat nucleus of the solitary tract (NTS), we induced c-Fos gene expression in NTS neurons with leptin and CCK. We focused on NTS catecholamine neurons because these cells have been implicated in the feeding response to CCK. Hindbrain sections from rats that received CCK with or without leptin pretreatment were immunostained for c-Fos and tyrosine hydroxylase (TH) by a double immunofluorescence procedure. Leptin pretreatment increased the number of NTS cells expressing c-Fos-like immunoreactivity (cFLI) 3-fold relative to CCK alone, but the number of TH-positive cells with cFLI was increased 6-fold. Next, cells detected by immunofluorescence for TH were collected by laser capture microdissection and pooled for real-time quantitative PCR of c-Fos mRNA. Here, neither le0ptin nor CCK alone affected the relative amount of mRNA in the TH cell-enriched samples, but leptin plus CCK substantially increased c-Fos mRNA content. These histochemical findings identify hindbrain catecholamine cells as potential mediators of the interaction between leptin and CCK.     

Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

Background  

The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.     

PMA-qPCR法检测冷冻基质中非可培养状态(VBNC)副溶血性弧菌

【背景】副溶血性弧菌为冰鲜产品及肉制品中常见的污染微生物,致病性强,危害严重,出入境运输及加工的肉食品常采取冷冻冷藏的处理手段来防止微生物污染及生长,以保持食物新鲜。而残留的部分副溶血性弧菌会进入活的非可培养状态(Viable but non-culturable state,VBNC),从而构成潜在的风险隐患。【目的】建立可用于冷冻食品中VBNC副溶血性弧菌的快速检测方法,并探讨其适用性。【方法】将大西洋鲑鱼1:10匀浆,加入终浓度为6.6×10~5 CFU/mL的副溶血性弧菌,-20°C分别诱导10、20、30和50 d。建立实时荧光PCR技术(qPCR)方法,测定其特异性、灵敏度及稳定性。利用PMA-qPCR法对不同冷冻时期样品中的副溶血性弧菌进行检测,同时与qPCR、平板培养法进行比较。【结果】建立的qPCR方法特异性好,与其他阴性参考株无交叉反应;灵敏度高,检测限为19.8 CFU/mL;重复性好,C_q值的变异系数(CV)均在1.5%以下;标准曲线为y=-3.272x+45.310,线性回归系数R~2为0.996,定量范围为1×10~2–1×10~9 CFU/mL。在低温诱导10-50 d后,qPCR法的C_q值在26.32-27.34之间,与诱导前相比几乎没有变化;叠氮溴化丙锭(PMA)-qPCR法的C_q值则从诱导前的26.43逐步上升到38.84,呈现明显的上升趋势,表明死菌的数量在显著上升。经过比较及统计,PMA-qPCR检测的活菌数均高于平板培养法测出的数量,差异显著(P0.05)。【结论】PMA-qPCR特异性及灵敏度高,能有效抑制对死菌的扩增,同时能克服传统平板培养法对VBNC的漏检缺陷,可方便、快捷地用于冷冻食品中受损致病微生物,尤其是进入VBNC状态的细菌检测。     

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

A TaqMan quantitative real-time PCR detection system was developed to examine transgene copy number in cotton. GhUBC1, a gene validated to be present as a single copy per haploid Gossypium hirsutum genome, was used as the endogenous reference to estimate copy number of GFP and selection marker NPTII in 28 T0 plants. This system was found to be more accurate than genomic Southern blot hybridization and could effectively tell homozygotes from heterozygotes in a T1 transgenic cotton population. Therefore it is suitable for efficient and cost effective early screening of transgenic seedlings and identifying transgene homozygotes in segregation populations.     

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.     

Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species

The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 degrees C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.