DNA Extraction Procedure Research Articles

The Chemistry behind DNA Isolation from Orange Juice and Detection of 16S rDNA of Candidatus Liberibacter asiaticus by qPCR

The current standard to diagnose Huanglongbing (HLB) for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and apply quantitative real-time PCR (qPCR) test to detect 16S rDNA of Candidatus Liberibacter asiaticus (CLas), the putative causal pathogen. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high pectin concentration, low pH and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice, and detection of CLas by qPCR. Homogenization using a sonicator increased DNA extraction by 86%, and stabilized quantification of 16S rDNA in comparison to mortar and pestle extraction, which showed wide variability of Ct values of 16S rDNA. Orange juice is rich in pectin, which has similar physiochemical features to DNA: soluble in water and precipitates in ethanol/isopropanol solutions. Thus, it is difficult to separate the DNA from pectin. However, DNA was successfully extracted by adding pectinase to hydrolyze the pectin. Without going through an elution column, the amplification of plant and microbial DNA in orange juice samples was inhibited by an unknown compound. Thus application of an elution column successfully eliminated the inhibitor. To eliminate errors caused by different methods of sampling, DNA extraction and qPCR procedures, Ct of a cytochrome oxidase (COX) to represent citrus plant DNA was detected as a reference, and a relative unit, ΔCt16S rDNA-COX was introduced to express the relative CLas population.

Transgene detection by digital droplet PCR.

Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

First Report of 16SrII-D Phytoplasma 'Candidatus Phytoplasma aurantifolia' Associated with Mung Bean Phyllody in Andhra Pradesh, India.

Mung bean (Vigna radiata (L.) R. Wilczek) is an important edible legume grown in Asia, particularly in the Indian subcontinent, where it is used for human and animal consumption. In September 2013, 10% of a group of 90 mung bean breeding lines in experimental plots of S. V. Agricultural College, Tirupati, Andhra Pradesh, India, exhibited symptoms typical of a phytoplasma infection, including stunting, extensive proliferation of branches, reduction in leaf size, phyllody, and longitudinal splitting of green pods followed by germination of green seeds producing small plants. These symptoms have been associated with mung bean phyllody (1,3). While the severity of infection varied within each line, on average, 20% of symptomatic plants did not produce seeds at all. Leaf samples from two symptomatic plants and one asymptomatic plant were collected and DNA was extracted from leaves following a CTAB DNA extraction procedure (2). Direct PCR and nested PCR assay was performed using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (4). Both of the symptomatic samples produced 1.8 kb and 1.2 kb size amplicons after direct and nested PCR cycles, respectively. No amplicons were produced with DNA from the asymptomatic sample. Nested PCR products (1.2 kb) from the two symptomatic samples corresponding to the F2nR2 region of 16S rDNA were directly sequenced on an ABI 3730 XL automated sequencer at McLab sequencing services (McLab, CA). Both samples were 100% identical and the representative sequence was designated as APMBP and deposited in GenBank with the accession number KF811205. BLAST analysis revealed a 100% sequence identity with 16SrII group 'Candidatus Phytoplasma aurantifolia' phytoplasmas that include sesame phyllody phytoplasmas isolated from sesame and tomato in India (KF429485 and JX104335, respectively). Subgroup identification was performed using the iPhyClassifier online tool (5). The samples were identified as 16SrII-D subgroup phytoplasma based on their 100% identity to the reference strain (GenBank Accession No. Y10097) and virtual RFLP profiles. Phylogenetic analysis based on the F2nR2 sequences with the representative sequences were placed the APMBP in a single distinct cluster with the 16SrII-D reference strain Y10097. Although occurrence of phyllody on mung bean in India was first reported in 1988 (3), the report was based on the appearance of symptoms. This pathogen was recently reported as associated with mung bean phyllody in Pakistan (1). To our knowledge, this is the first report of 'Ca. P. aurantifolia' strain infecting mung bean in India. Phytoplasmas belonging to subgroup 16SrII-D are known to have a wide host range, including chickpea, peanuts, sesame, and tomato, which are commonly grown in this region. Mung bean plants infected early failed to produce normal seeds indicating of the potential of this 16SrII-D phytoplasma to become a production constraint for mung bean and other host crops in the area.

An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region.

The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method.

Meta‐barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard.

Detection of Candida vulvovaginitis in Clinical Samples ; Using Direct Polymerase Chain Reaction Without DNA Extraction

Background: Vulvovaginal candidiasis (VVC) is a common disease which infects women. The current study investigated the performance of direct sample polymerase chain reaction (DS-PCR) method to detect Candida spp. in clinical samples of vulvovaginitisto compare the results to those of standard microbiological laboratory methods. Objectives: The current study aimed to further simplify the DNA extraction procedure, and shorten the time required for isolation and identification by using direct PCR to identify Candida vulvovaginitis without DNA extraction from samples or colonies. Patients and Methods: In the current study, totally 150 sexually active women participated. Vaginal discharge samples were collected using two sterile Dacron swabs that were immediately placed in two tubes each containing 1 mL of distilled water. One of the tubes was used for conventional culture methods whereas the other one was used for DS-PCR without DNA extraction. The number of yeast cells in each sample was counted. Results: The results showed that out of the 150 samples, 55 were positive and 63 samples were negative by both methods, and 32 samples were positive using the culture method, but negative by DS-PCR. All positive DS-PCR samples had > 10 7 yeast or conidia cells/mL. The sensitivity and specificity of DS-PCR were calculated as 63.2% and 100%, respectively. Conclusions: Direct sample PCR has the potential to rapidly and accurately diagnose Candida vulvovaginitis in patients, especially if sufficient samples are obtained.

Diverse DNA extraction methods and PCR primers for detection of Huanglongbing-associated bacteria from roots of ‘Valencia’ sweet orange on sour orange rootstock

Fibrous root samples from Huanglongbing symptomatic (S) and asymptomatic (AS) ‘Valencia’ sweet orange trees (Citrus sinensis) grafted onto sour orange (C. aurantium) rootstock were analyzed using different DNA extraction procedures and primers to detect Candidatus Liberibacter asiaticus. Root sampling procedure was modified from the conventional use of soil probe to reduce fatigue, and to facilitate sample collection. DNA extraction kits including Qiagen DNeasy, Mo Bio PowerSoil and PowerPlant generated root DNA suitable for polymerase chain reaction (PCR) diagnostic assays. Quantitative PCR (qPCR) using HLBaspr primers and probe on root DNA extracts from a total of 206 samples from 60 AS trees showed unreliable results for the presence of Ca. L. asiaticus. Except for one sample, all the remaining samples gave negative qPCR result using LJ900fpr primers and probe. Further analysis of roots samples from S and AS trees indicate that HLBaspr is not suitable for Ca. L. asiaticus diagnosis in root samples. Leaf samples from all 60 AS trees showed negative qPCR result with HLBaspr. Conventional PCR (cPCR) using OI1/OI2c primers on 10 randomly selected root samples, from the 206 samples, produced non-specific amplification products which were easily distinguished from products specific to Ca. L. asiaticus seen in the positive control. The same root samples did not produce any amplification product in cPCR using A2/J5 primers. Any of the above mentioned cPCR or qPCR assays could accurately detect Ca. L. asiaticus in S leaf samples of Ca. L. asiaticus-infected trees. Root samples from 10 (5 S and 5 AS) additional trees analyzed by cPCR using A2/J5 and Las606/LSS primers and qPCR using LJ900fpr and CQULA (CQULA04F/CQULA04R, and TaqMan probe CQULAP10) clearly showed that all these primers are efficient in detecting Ca. L. asiaticus in root samples. There was no difference in frequency and consistency of Ca. L. asiaticus detection for root samples collected near the tree trunk compared to the roots collected about 1.8m from the trunk. Ca. L. americanus was not detected in either root or leaf samples.

Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei

Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable identification of transposon insertion sites in a L. casei mutant library of 9250 mutants. DNA extraction procedure based on silica membranes in 96-column format was optimized to obtain genomic DNA from a large number of mutants. Then reliable direct genomic sequencing was improved to fit the obtained genomic DNA extracts. Using this procedure, readable and identifiable sequences were obtained for 87% of the L. casei mutants. This method extends the applications of a library of this type, reduces the number of insertions needed to be screened, and allows selection of specific mutants from an arrayed and stored mutant library. This method is applicable to any already existing mutant library (obtained by transposon or insertional mutagenesis) and could be useful for other bacterial species, especially for highly lysis-resistant bacteria species such as lactic acid bacteria.

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.

The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons.

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

An Alternative and Rapid Method for the Extraction of Nucleic Acids from Ixodid Ticks by Potassium Acetate Procedure

Four variants of the potassium acetate procedure for DNA extraction from ixodid ticks at different stage of their life cycles were evaluated and compared with phenol-chloroform and ammonium hydroxide methods. The most rapid and most efficient variant was validated in the DNA extraction procedure from the engorged ticks collected from bovine, canine as well as from house ticks for the screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. The ammonium hydroxide procedure was used for non-engorged ticks. All the variants were efficient and allowed obtaining PCR-quality material according to the specific amplification of 16S rRNA gene fragment of the original tick. DNA extracted from the ticks under the study was tested by multiplex PCR for the screening of tick-borne pathogens. Anaplasma spp. and Babesia spp. amplification products were obtained from 29/48 extracts. Ammonium hydroxide protocol was not efficient for two extracts. Detection of amplification products from the PCR indicated that DNA had been successfully extracted. The potassium acetate procedure could be an alternative, rapid, and reliable method for DNA extraction from the ixodid ticks, mainly for poorly-resourced laboratories.

An Alternative and Rapid Method for the Extraction of Nucleic Acids from Ixodid Ticks by Potassium Acetate Procedure

Four variants of the potassium acetate procedure for DNA extraction from ixodid ticks at different stage of their life cycles were evaluated and compared with phenol-chloroform and ammonium hydroxide methods. The most rapid and most efficient variant was validated in the DNA extraction procedure from the engorged ticks collected from bovine, canine as well as from house ticks for the screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. The ammonium hydroxide procedure was used for non-engorged ticks. All the variants were efficient and allowed obtaining PCR-quality material according to the specific amplification of 16S rRNA gene fragment of the original tick. DNA extracted from the ticks under the study was tested by multiplex PCR for the screening of tick-borne pathogens. Anaplasma spp. and Babesia spp. amplification products were obtained from 29/48 extracts. Ammonium hydroxide protocol was not efficient for two extracts. Detection of amplification products from the PCR indicated that DNA had been successfully extracted. The potassium acetate procedure could be an alternative, rapid, and reliable method for DNA extraction from the ixodid ticks, mainly for poorly-resourced laboratories.

First Report of QOI-insensitive Powdery Mildew (Erysiphe polygoni) on Sugar Beet in the United States.

The $2.1 billion United States sugar beet (Beta vulgaris L.) industry is the primary provider of domestic sucrose. Sugar beet powdery mildew is caused by Erysiphe polygoni DC and occurs principally in sugar beet growing regions in the western United States. In these regions, the quinone outside inhibitor (QOI) fungicides pyraclostrobin (Headline, BASF, NC) and trifloxystrobin (Gem, Bayer Crop Science, NC) have been important tools to manage powdery mildew since registration in 2002 and 2005, respectively. However, researchers in Idaho reported poor disease management despite QOI application starting in 2011. In 2013, a research plot near Parma, ID, containing natural powdery mildew infection received treatments of pyraclostrobin, trifloxystrobin, or was untreated (control). Since there was no significant reduction in disease levels between QOI-treated blocks and untreated control blocks, experiments were conducted to clone a partial fragment of the E. polygoni cytochrome b (cytb) gene to gain insight into the molecular basis of QOI resistance in this pathosystem. The primers MDB-920 (5'-CACATCGGAAGAGGTTTATA-3') and MDB-922 (5'-GGTATAGATCTTAATATAGCATAG-3') were designed based on consensus sequences of several fungal cytb genes obtained from GenBank (data not presented) and used to amplify a 575-bp fragment of the E. polygoni cytb gene using DNA isolated from 12 infected leaf samples collected in September 2013 from the Parma research plot. Each sample consisted of three leaves harvested from three plants (one leaf per plant) in an experimental block. All DNA extraction, PCR, and sequencing procedures were as described previously (1). PCR products derived from six QOI-treated samples and six untreated samples were sequenced directly. Without exception, all QOI-treated samples harbored a point mutation at nucleotide position 143 that encoded a G143A mutation compared with cytb sequence from untreated samples. The two identified cytb haplotypes have been deposited in GenBank under accession numbers KF925325 and KF925326. This is the first report of QOI resistance and the associated cytb G143A mutation in E. polygoni. The G143A mutation has been reported in most QOI-resistant pathogens to date (2). When the G143A mutation dominates in a pathogen population, there is a consistent association with a loss of disease management despite QOI application (3). Careful monitoring and judicious use of QOI fungicides will be necessary to ensure QOI fungicides remain efficacious for sugar beet powdery mildew management in the United States.

DNA isolation by Chelex-100: an efficient approach to consider in leptospirosis early stages

Objective: To compare the value of leptospiral DNA extraction procedures from clinical samples for the early diagnosis of leptospirosis. Methods: Three DNA extraction procedures were applied for microbiological analysis, results of QIAmp DNA mini kit (QIAGEN, Germany), CLART HPV kit (GENOMICA, Spain) and Chelex-100 assay were compared concerning extraction efficiency, DNA purity and DNA suitability for amplification by specific polymerase chain reaction for pathogenic leptospires from blood, plasma and serum artificially infected. Results: The comparison of extraction methods highlighted the efficiency of Chelex-100 and QIAmp DNA mini kit. Chelex-100 achieved the isolation of the highest concentration of leptospiral DNA from the culture and the spiked samples, with acceptable purities and without inhibitors to PCR. Conclusions: Chelex-100 assay is a rapid and effective approach for DNA isolation in clinical samples having pathogenic leptospires and it could be useful in the early diagnosis of leptospirosis.

Evaluation of techniques for human bone decalcification and amplification using sixteen STR markers

Efficient DNA extraction procedures, as well as accurate DNA amplification, are critical steps involved in the process of successful DNA analysis of skeletal samples. Unfortunately, at present there is no infallible method to recover DNA from highly degraded samples due to variations in DNA yield from larger bone fragments, which may be attributed to heterogeneity within bones. We evaluated two different protocols for bone decalcification in the DNA extraction procedure for bones. This study is important for analysis of challenging forensic samples.

Impact of fruit processing methods on DNA extraction from transgenic frozen banana products

Isolation of high yield and quality of genomic DNA is paramount for ensuring confidence in molecular analyzing food. This study evaluated five different DNA extraction procedures based on laboratory protocols and commercial kits for their efficiency and also assessed the impact of processing procedures on the DNA degradation in various frozen banana products such as frozen-sliced banana, frozen banana puree, frozen plantain banana, and freeze-dried banana powder. The purity and concentration of DNA obtained from the samples were evaluated by spectrophotometric and gel electrophoresis analysis. Of the various extraction procedures, we applied modified protocols that allow for effective extraction of DNA from banana. This protocol derived from traditional cetyltrimethylammonium bromide method with modifications that allowed removing unwanted polyphenols and polysaccharides. The results confirm that a profound impact on DNA degradation was seen during the processing of banana products, DNA could still be reliably quantified by real-time PCR. Furthermore, frying resulted in further degradations and drying resulted in the most severe changes. It was noted that in different frozen banana products, nopaline synthase fragment is most stable followed by cauliflower mosaic virus 35S and NPTII gene.

DNA extraction protocols for molecular studies of Asiatic black bears

Abstract Optimization of DNA extraction and amplification techniques increases the reliability of genetic studies used to estimate population size and assess genetic diversity, particularly for non-invasively collected samples. We evaluated the effectiveness of 4 DNA extraction procedures—the Roboscreen and Qiagen kit methods and the Organic and Chelex methods, as applied to invasively and non-invasively collected samples from Asiatic black bears (Ursus thibetanus). We assessed effectiveness based on quantity and purity of the extracts by method, and their ability to amplify genomic DNA via polymerase chain reaction (PCR) amplification of a single simple sequence repeat (SSR) marker. For each method, the greatest average DNA quantity and purity index (86.25%) was from blood, while muscle showed greatest DNA quantity and purity index using the Qiagen DNA extraction method. The Roboscreen kit and Chelex method provided a 57% and 58% PCR success rate, respectively, for hairs, demonstrating the value of such ...

Diagnostic accuracy of the PURE-LAMP test for pulmonary tuberculosis at the county-level laboratory in China.

BackgroundEarly and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China.Methodology/Principal FindingsFrom April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture.Conclusions/SignificanceThe study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.

Modified pretreatment method for total microbial DNA extraction from contaminated river sediment

Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatment method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters (i.e., reagent solution, reaction duration, and temperature). The pretreatment procedure involves washing the river sediment sample for three times with a solution containing 0.1 mol·L−1 ethylene diamine tetraacetic acid (EDTA), 0.1 mol·L−1 Tris (pH 8.0), 1.5 mol·L−1 NaCl, 0.1 mol·L−1 NaH2PO4, and Na2HPO4 at 65°C with 180 r·min−1 for 15 min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatment, without pretreatment, and with using a commercial PowerSoil™ DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the PowerSoil™ DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample (0.25 g or even lower). The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes.

Diagnosis of Mycobacterium bovis in Captive Sloth Bears (Melursus ursinus) by Polymerase Chain reaction

Diagnosis of mycobacterial infection is paramount important from the public health perspective since treatment and control measures are very significant, particularly in captive animals. In this diagnostic study of Mycobacterium bovis infection in sloth bears (Melursus ursinus), polymerase chain reaction (PCR) had been used with the primer sequence of pncA-8 (5′-GGTTGGGTGGCCGCCGGTCAG-3′) and pncA-11 (5′-GCTTTGCGGCGAGCGCTCCA-3′) that were specific for M. bovis pncA gene. Forty-two fresh faecal samples were collected randomly from the apparently healthy sloth bears maintained in captive conditions. The DNA extraction procedure was done as per the manufacturer’s protocol and further subjected to amplification. The amplification profile includes respectively: initial heating of the samples for 5 min at 94 °C, annealing at 55 °C for 1 min, primer extension at 72 °C for 1 min and final elongation step for 10 min at 72 °C. Out of 57 samples, 5 samples were yielded on expected amplified PCR product size of 744 bp when electrophoresed in 1.5 % agarose gel. A positive control of M. bovis DNA procured from Tuberculosis Research Centre and a negative control from a healthy bovine sample were used. These results demonstrated that PCR test will increase the effectiveness of laboratory diagnosis to detect and identifying the M. bovis in captive wild animals.