三种粪便总DNA提取方法的比较

目的比较不同粪便总DNA提取方法对肠道菌群多样性研究的影响。方法采用Bead beating法、化学裂解法和QIAamp DNA Stool Mini Kit提取同一份人粪便样品的总DNA,对比3种方法的DNA得率和16S rRNA基因V3区的变性梯度凝胶电泳（DGGE）图谱。结果Bead beating法的DNA得率约是其他2种方法的2倍;3种方法得到的DGGE图谱的Dice相似性为60％～70％,2条优势条带只出现在Bead beating法图谱中。在2～5min的Bead beating法击打时间里,DNA得率随击打时间的延长有一定的增加,但DGGE图谱无显著变化。结论不同的DNA提取方法会影响菌群的多样性分析。比较其他2种方法,Bead beating的裂解效率更高,能够检测到更多种类的细菌,更合适肠道菌群组成的分子研究。     

沼气池污泥微生物总DNA提取方法的比较

沼气发酵系统是一个复杂的生态系统,其污泥微生物超过99%是不可培养的。为了优化沼气池纤维素的转化效率、沼气的产率和开展污泥微生物多样性研究,本研究采用化学裂解法、溶菌酶裂解法和QIAampDNA Stool Mini Kit提取了沼气池污泥样品中微生物的总DNA,对三种方法的DNA得率、纯度、大片段提取效果以及是否含有PCR反应抑制剂进行了研究,最后对16S rRNA基因V3区的扩增产物进行了PCR变性梯度凝胶电泳(PCR-DGGE)分析。与化学裂解法和QIAamp DNA Stool Mini Kit法相比,溶菌酶裂解法得到的DNA量大、片段长、片段分布广、PCR扩增效率高;同时PCR-DGGE图谱显示,溶菌酶裂解法可更好地展示沼气池污泥中微生物的多样性。该结果为进一步提高沼气池中纤维素的转化效率和沼气生产优势菌种的质和量打下了一定的前期基础。     

蝗虫肠道微生物总DNA提取方法的比较

采用Bead beating法和QIAamp DNA stool mini kit法提取蝗虫肠道微生物总DNA,并对2种方法提取DNA的得率、完整性以及16SrRNA基因扩增产物的变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)图谱等进行综合比较。结果表明,Bead beating法提取DNA的得率显著高于QIAamp DNA stool mini kit法(P=0.042),而QIAamp DNA stool mini kit法提取DNA片段更完整。PCR-DGGE检测微生物多样性结果显示,QIAamp DNA stool mini kit法提取DNA所代表的微生物群落多样性略高于Bead beating法,但Mann-Whitley统计学检验表明用2种方法检测蝗虫肠道微生物多样性无显著差异(P=0.17)。因此在蝗虫肠道微生物群落多样性的检测中QIAamp DNA stool mini kit法具一定的优势,而Bead beating法同样适用。     

Giardia intestinalis: DNA extraction approaches to improve PCR results

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (101 cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 102 spores and 75 % for 101 spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.     

Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C_T) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10⁴ colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.     

Evaluation of five commercial nucleic acid extraction kits for their ability to inactivate Bacillus anthracis spores and comparison of DNA yields from spores and spiked environmental samples

This study evaluated five commercial extraction kits for their ability to recover DNA from Bacillus anthracis spores and spiked environmental samples. The kits evaluated represent the major types of methodologies which are commercially available for DNA or total nucleic acid extraction, and included the ChargeSwitch gDNA Mini Bacteria Kit, NucliSens Isolation Kit, Puregene Genomic DNA Purification Kit, QIAamp DNA Blood Mini Kit, and the UltraClean Microbial DNA Isolation Kit. Extraction methods were performed using the spores of eight virulent strains of B. anthracis. Viability testing of nucleic acid extracts showed that the UltraClean kit was the most efficient at depleting samples of live B. anthracis spores. TaqMan real-time PCR analysis revealed that the NucliSens, QIAamp and UltraClean kits yielded the best level of detection from spore suspensions. Comparisons of processed samples from spiked swabs and three powder types indicated that DNA extraction using the UltraClean kit resulted in the most consistently positive results and the lowest limit of detection. This study demonstrated that different nucleic extraction methodologies, represented here by various commercial extraction kits, differ in their ability to inactivate live B. anthracis spores as well as DNA yield and purity. In addition, the extraction method used can influence the sensitivity of real-time PCR assays for B. anthracis.     

体重差异双胎新生儿 粪便样本DNA提取的优化及分析

摘要：目的 优化新生儿粪便样本DNA提取方法,提取及分析体重差异双胎新生儿粪便样本DNA。方法 从7种DNA提取试剂盒方法中选择对成人粪便样本DNA提取效果最佳的QIAamp DNA Stool Mini Kit法为基准方法,通过钢珠打断前处理、DNA吸附柱收集全部裂解液上清和洗脱液重复洗脱的优化,建立了用于新生儿粪便样本DNA的提取方法。结果 该优化方法用于婴儿（出生1个月）粪便样本,结果显示DNA提取浓度平均提高了2.4倍。用于48对双胞胎新生儿出生第1天和第3天粪便样本DNA的提取,经酶标仪及PCR扩增检测,结果显示出生第1天粪便样本DNA提取率为32%,出生3天提取率达83%。RT-PCR显示新生儿第1天到第3天肠道微生物量呈现增长趋势。结论 优化的QIAamp DNA Stool Mini Kit法适用于新生儿粪便样本DNA的快速提取,为后续扩增子高通量测序和研究体重差异双胎新生儿肠道菌群构成规律奠定基础。     

Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota

Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 degrees C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5-8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.     

PCR-based detection of genetically modified soybean and maize in raw and highly processed foodstuffs

The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor in the production of meaningful results. In this study, we present two procedures that enable an efficient isolation of trace amounts of genetic material from both raw and highly processed foodstuffs. We show that for optimal, PCR-ready DNA purification from highly processed foodstuffs and PCR inhibitor-rich substances--such as cocoa-containing products--adapted protocols for the QIAGEN QIAamp DNA Stool Mini Kit can be utilized. For complete DNA isolation from raw foodstuffs, a protocol using the DNeasy Plant Mini Kit is presented.     

DNA extraction method affects microbial community profiles from soils and sediment

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.     

Improved extraction of PCR-quality community DNA from digesta and fecal samples

Several DNA extraction methods have been reported for use with digesta or fecal samples, but problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances. Here we report a modified method to extract PCR-quality microbial community DNA from these types of samples, which employs bead beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, and with subsequent DNA purification by QIAamp columns [referred to as repeated bead beating plus column (RBB + C) method]. The RBB + C method resulted in a 1.5- to 6-fold increase in DNA yield when compared to three other widely used methods. The community DNA prepared with the RBB + C method was also free of inhibitory substances and resulted in improved denaturing gradient gel electrophoresis (DGGE) profiles, which is indicative of a more complete lysis and representation of microbial diversity present in such samples.     

Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm

The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.     

Development of a multiplex-PCR for rapid detection of the enteric pathogens Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli in porcine faeces

AIMS: To develop an assay to simultaneously detect Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig faeces. METHODS AND RESULTS: A multiplex-polymerase chain reaction (M-PCR) was designed to amplify a 655-base pair (bp) portion of the L. intracellularis 16S rRNA gene, a 354-bp portion of the B. hyodysenteriae NADH oxidase gene, and a 823-bp portion of the B. pilosicoli 16S rRNA gene. Specificity was assessed using 80 strains of Brachyspira spp. and 30 other enteric bacteria. Bacterial DNA was extracted from faeces using the QIAamp DNA Stool Mini Kit. The M-PCR was tested in parallel with culture and/or PCR on 192 faecal samples from eight piggeries. Faeces also were seeded with known cell concentrations of the three pathogenic species, and the limits of detection of the M-PCR tested. The M-PCR was specific, with limits of detection of 10(2)-10(3) cells of the respective species per gram of faeces. CONCLUSIONS: The M-PCR is a rapid, sensitive and specific test for detecting three important enteric bacterial pathogens of pigs. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of a new diagnostic M-PCR will allow rapid detection and control of three key porcine enteric pathogens.     

两种核酸提取方法对小鼠诺如病毒RNA提取效能的比较

目的比较两种核酸提取方法对小鼠诺如病毒RNA的提取效能。方法用Trizol提取法和QIAamp Vira lRNA Min iKit提取法分别提取感染小鼠诺如病毒（Murine Norovirus,MNV）的小鼠小肠组织样品RNA和细胞培养物RNA,测定RNA浓度;用MNV特异的引物对分离的核酸样品进行一步法RT—PCR扩增。结果Trizol提取法提取小肠组织的RNA浓度高于QIAamp Viral RNA Mini Kit提取法;QIAamp Viral RNA Mini Kit提取得到的细胞培养物RNA浓度高于Trizol提取法。经QIAamp Viral RNA Mini Kit提取的两种核酸样品均能扩增出特异条带,而Trizol提取的核酸样品未见特异条带。结论在MNV的检测中,QIAampViralRNAKit更适合组织样品中MNV病毒核酸的提取。