共查询到20条相似文献,搜索用时 15 毫秒
1.
Sivashanmugam Parthipan Sellappan Selvaraju Lakshminarayana Somashekar Atul P. Kolte Arunachalam Arangasamy Janivara Parameswaraiah Ravindra 《Analytical biochemistry》2015
Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at −80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy + TRIzol and PureLink + TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800–900 ng/30–40 × 106) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50–2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa. 相似文献
2.
Biomolecular hybrids of a conducting polymer [poly(o-methoxy aniline) (POMA)] and RNA are prepared at the three different compositions by mixing aqueous solutions of diethyl, 2-hydroxy ethyl, ammonium salt of RNA (type IX from Torula Yeast) and POMA (ES, emeraldine salt; doping level [Cl]/[N] = 0.52). A slow increase of pH up to 30 h of aging occurs in the mixture till it levels up. The TEM micrographs indicate a fibrillar network structure in all the hybrid compositions (POMA: RNA = 1:3, 1:1, 3:1, by weight). In the complexes three types of supramolecular interactions, viz. (i) electrostatic, (ii) H-bonding and (iii) π–π interactions, are evident from the FTIR spectroscopy. The CD spectra indicate a small distortion of A-RNA conformation towards its B form during the hybrid formation. Time and temperature dependent UV–vis spectral studies indicate a slow red shift of the π-band to polaron band transition peak (λmax) for the uncoiling of the POMA (P) chain on the RNA (R) surface. The repulsive interaction between the radical cations of POMA (ES) absorbed on the RNA surface is attributed to the conformational change causing the uncoiling of POMA chain. UV–vis spectral study indicates that the uncoiling and attachment of POMA on RNA surface is much faster than that on DNA (D). In POMA–RNA–DNA (PRD) hybrid solutions slower red shift of λmax indicates more disordered array of the phosphate groups than that in PR and PD systems. The conductivity values of the PR hybrids (10− 6 S/cm− 1) are three orders higher than that of RNA, rendering the PR hybrids to be useful for fabricating good biosensors. In the PRD hybrids conductivity decreases by two orders than those of PR and PD hybrids suggesting a disorder arrangement of POMA chains in the PRD hybrids. The I–V characteristic curves of the PR and PRD hybrids indicate a semiconducting nature of the hybrids. 相似文献
3.
This study investigated the effects of light (visible light - 5800 lux, 24 h) or dark regime and aerobic or anaerobic condition on the decay of added oxolinic acid (OA) at 5, 10 and 20 mg L−1 in eel pond sediment. An asymptotic decaying exponential model Ct = Cmin + Co × exp (−k × t) was used to facilitate quantitative approach to OA transformation, where Ct is the concentration of OA after t days, Cmin the estimated level-off concentration of OA residue, Co the concentration of added OA and k the decaying coefficient. OA decayed faster under light (Cmin = 4.6 mg L−1) than under dark (Cmin = 7.8 mg L−1) and also decayed faster under aerobic (Cmin = 4.0 mg L−1) than under anaerobic condition (Cmin = 8.5 mg L−1). Cmin increased with Co. Sundrying and tilling eel pond bottom should be able to reduce OA residue significantly. 相似文献
4.
5.
Zhenyu Zhu Liang Zhao Xiaofan Liu Jun Chen Hai Zhang Guoqing Zhang Yifeng Chai 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(24):2184-2190
The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C18 column (150 mm × 4.6 mm, i.d.: 5 μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156 μg mL−1 for baicalin and 0.08832 to 4.416 μg mL−1 for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155 μg mL−1 for baicalin and 0.04416 μg mL−1 for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t1/2 of baicalin was 3.60 ± 0.90 and 5.64 ± 1.67, the Cmax1 was 1.64 ± 0.99 and 5.66 ± 2.02, the tmax1 was 0.13 ± 0.05 and 0.20 ± 0.07, the Cmax2 was 2.43 ± 0.46 and 3.18 ± 1.66, and the tmax2 were 6.40 ± 1.67 and 5.66 ± 2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t1/2 of wogonoside was 4.97 ± 1.68 and 7.71 ± 1.55, the Cmax1 was 1.39 ± 0.83 and 1.45 ± 0.37, the tmax1 was 0.21 ± 0.20 and 0.17 ± 0.01, the Cmax2 was 1.90 ± 0.55 and 1.42 ± 0.70, and the tmax2 was 5.60 ± 1.67 and 5.20 ± 1.79, respectively. A significant difference (p < 0.05) was observed for t1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased. 相似文献
6.
María José León Cristina Sánchez-Porro Rafael R. de la Haba Inmaculada Llamas Antonio Ventosa 《Systematic and applied microbiology》2014
Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C18:1ω7c/C18:1ω6c, C16:0, and C16:1ω7c/C16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18T (= CCM 8464 = CECT 8192T = IBRC-M 10767T = LMG 27461T). 相似文献
7.
Xue Hai Ju Ying Shi Na Liu Dong Mei Guo Xi Cui 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(22):1982-1986
In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X2) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: Cmax, 1484.55 ± 285.05 ng/ml; Tmax, 0.81 ± 0.16 h; t1/2α, 3.78 ± 2.33 h; t1/2β, 6.06 ± 3.20 h; t1/2Ka, 0.18 ± 0.53 h; K12, 0.18 ± 0.41/h; K21, 0.20 ± 0.16/h; K10, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS. 相似文献
8.
Weixuan Yu Carla Neckles Andrew Chang Gopal Reddy Bommineni Lauren Spagnuolo Zhuo Zhang Nina Liu Christina Lai James Truglio Peter J. Tonge 《Analytical biochemistry》2015
The classical methods for quantifying drug–target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-acyl carrier protein (enoyl-ACP) reductase as a model system, we developed a Penefsky column-based method for direct measurement of tR, where the off-rate of the drug was determined with radiolabeled [adenylate-32P]NAD(P+) cofactor. In total, 23 FabI inhibitors were analyzed, and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percentage drug–target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady-state kinetic methods for compounds with tR values of 10 to 100 min. In addition, we were able to identify seven long tR inhibitors (100–1500 min) and to accurately determine their tR values. The method was then used to measure tR as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in tR was observed when the temperature was increased from 25 to 37 °C. 相似文献
9.
Hongjun Xia Xiaodan WangLi Li Shengjia WangChangchuan Guo Yao LiuLushan Yu Huidi JiangSu Zeng 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(28):2701-2706
A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C18 column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r2) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters Cmax, Tmax, t1/2, AUC0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively. 相似文献
10.
Julia Firsova Nina Doronina Elke Lang Cathrin Spröer Stéphane Vuilleumier Yuri Trotsenko 《Systematic and applied microbiology》2009
A novel aerobic facultative methylotroph was isolated from contaminated soil. The organism (strain DM16) is a Gram-negative asporogenous non-motile curved rod multiplying by binary fission. Cells are neutrophilic and mesophilic. This strain utilized dichloromethane, methanol, formate and formaldehyde along with a variety of polycarbon compounds. Strain DM16 employs the ribulosebisphosphate pathway for C1 assimilation. The DNA G+C content is 64.5 mol%. The major ubiquinone is Q-10. The dominant cellular fatty acids are 18:1ω7c (58.6%), cyclo-19:0ω8c (34.8%) and 16:0 (3.2%). Sequencing of the 16S rRNA gene and DNA–DNA hybridization experiments clearly indicated that this methylotroph should be classified as a new species within genus Ancylobacter – Ancylobacter dichloromethanicus sp. nov. with the type strain DM16T (DSM 21507T=VKM B-2484T). 相似文献
11.
Lukasz Ponikiewski 《Inorganica chimica acta》2005,358(4):1322-1326
The insertion reaction of maleic anhydride into the Cu-O bond in [CuOtBu] produced the complexes [Cu2(CO2C2H2CO2tBu)4 · dme] (1), [Cu(CO2C2H2CO2tBu)2 · tmeda] (2) and [Cu2(CO2C2H2CO2tBu)2 · dppm]2 (3) (dme = 1,2-dimethoxyethane; tmeda = N1,N1,N2,N2-tetramethylethane-1,2-diamine; dppm = bis(diphenylphosphino)methane). This reaction represents a useful synthetic strategy for a range of functionalised Cu(I) and Cu(II) carboxylates. 相似文献
12.
13.
The reaction of halflanthanidocene aryloxides CpR′Ln(OArtBu,R)2 (Ln = Y, La, Lu; CpR′ = C5Me5, C4Me4H; R = H, Me) and halflanthanidocene alkoxides [(C5Me5)Ln(OCH2CMe3)2]2 (Ln = Y, Lu) with trimethylaluminum (TMA) was investigated. Monomeric CpR′Ln(OArtBu,R)2, derived from the ortho-tBu-substituted OC6H2tBu2-2,6-R-4 (R = H, Me) ligands, form mono(tetramethylaluminate) complexes CpR′Ln(OArtBu,R)(AlMe4) for the smaller lanthanide metal centers yttrium and lutetium. Such an [aryloxide] → [aluminate] ligand exchange was not observed at the larger lanthanum metal center. The mobility of the tetramethylaluminate ligands of complexes CpR′Ln(OArtBu,R)(AlMe4) (Ln = Y, Lu) was examined by variable-temperature (VT) 1H NMR spectroscopy, revealing two signals for bridging and terminal methyl groups at lower temperatures. The treatment of complexes CpR′Ln(OArtBu,R)(AlMe4) with donor solvent d8-THF gave CpR′Ln(OArtBu,R)(Me)(d8-THF)2 (Ln = Y, Lu) with terminal methyl groups, according to a donor-induced aluminate cleavage reaction. Dimeric [(C5Me5)Ln(OCH2CMe3)2]2 (Ln = Y, Lu) was synthesized from (C5Me5)Ln(NiPr2)2(THF) and reacted with two equivalents of TMA per Ln center to yield monomeric bis(TMA) adduct complexes (C5Me5)Ln(OCH2CMe3)2(AlMe3)2(Ln = Y, Lu). VT NMR spectroscopic studies confirmed a high mobility of the Ln(μ-OCH2CMe3)(μ-Me)AlMe2 moieties at an ambient temperature. Both bis(TMA) adduct complexes were characterized by X-ray structure analysis. 相似文献
14.
Synthesis and properties of fatty acid starch esters 总被引:3,自引:0,他引:3
Being completely bio-based, fatty acid starch esters (FASEs) are attractive materials that represent an alternative to crude oil-based plastics. In this study, two synthesis methods were compared in terms of their efficiency, toxicity and, especially, product solubility with starch laurate (C12) as model compound. Laurates (DS > 2) were obtained through transesterification of fatty acid vinylesters in DMSO or reaction with fatty acid chlorides in pyridine. The latter lead to higher DS-values in a shorter reaction time. But due to the much better solubility of the products compared to lauroyl chloride esterified ones, vinylester-transesterification was preferred to optimize reaction parameters, where reaction time could be shortened to 2 h. FASEs C6–C18 were also successfully prepared via transesterification. To determine the DS of the resulting starch laurates, the efficient ATR-IR method was compared with common methods (elementary analysis, 1H NMR). Molar masses (Mw) of the highly soluble starch laurates were analyzed using SEC-MALLS (THF). High recovery rates (>80%) attest to the outstanding solubility of products obtained through transesterification, caused by a slight disintegration during synthesis. Particle size distributions (DLS) demonstrated stable dissolutions in CHCl3 of vinyl laurate esterified – contrary to lauroyl chloride esterified starch. For all highly soluble FASEs (C6–C18), formation of concentrated solutions (10 wt%) is feasible. 相似文献
15.
Chi-Neng Lin Shu-Yii Wu Jian-Sheng Chang Jo-Shu Chang 《Bioresource technology》2009,100(13):3298-3301
Ethylene–vinyl acetate (EVA) copolymer was used to immobilize H2-producing sewage sludge for H2 production in a three-phase fluidized bed reactor (FBR). The FBR with an immobilized cell packing ratio of 10% (v/v) and a liquid recycle rate of 5 l/min (23% bed expansion) was optimal for dark H2 fermentation. The performance of the FBR reactor fed with sucrose-based synthetic medium was examined under various sucrose concentration (Cso) and hydraulic retention time (HRT). The best volumetric H2 production rate of 1.80 ± 0.02 H2 l/h/l occurred at Cso = 40 g COD/l and 2 h HRT, while the optimal H2 yield (4.26 ± 0.04 mol H2/mol sucrose) was obtained at Cso = 20 g COD/l and 6 h HRT. The H2 content in the biogas was stably maintained at 40% or above. The primary soluble metabolites were butyric acid and acetic acid, as both products together accounted for 74–83% of total soluble microbial products formed during dark H2 fermentation. 相似文献
16.
Valentina Bosello-Travain Marcus Conrad Giorgio Cozza Alessandro Negro Silvia Quartesan Monica Rossetto Antonella Roveri Stefano Toppo Fulvio Ursini Mattia Zaccarin Matilde Maiorino 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.Methods
Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.Results
Oxidation of the CP is fast (k+ 1 > 103 M− 1 s− 1), however the rate of reduction by GSH is slow (k′+ 2 = 12.6 M− 1 s− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+ 1 > 103 M− 1 s− 1), but not by Trx. By surface plasmon resonance analysis, a KD = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.Conclusions
GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.General significance
In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. 相似文献17.
18.
The RNA of Balbiani ring BR2 of polytene chromosomes from Chironomus thummi salivary glands was microisolated and reassociated in the presence of an excess of total larval DNA. BR2 RNA reacts as a single component with a C0t1/2 of 8.6. Ribosomal precursor RNA from microisolated nucleoli reassociates under identical conditions with a C0t1/2 of 12.3. These C0t1/2-values suggest repetition frequencies in the range of 35 and 50 for ribosomal DNA and Balbiani ring 2 DNA, respectively. The data presented here favour the view that the gene for BR2 RNA of C. thummi is internally repeated and contains only one type of DNA sequence. 相似文献
19.
Nina V. Doronina Maria N. Poroshina Elena N. Kaparullina Vladimir A. Ezhov Yuri A. Trotsenko 《Systematic and applied microbiology》2013
Two newly isolated halotolerant obligately methylotrophic bacteria (strains C2T and SK12T) with the serine pathway of C1 assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, non-motile rods, forming rosettes, multiplying by binary fission. Mesophilic and neutrophilic, accumulate intracellularly compatible solute ectoine and poly-β-hydroxybutyrate. The novel strains are able to grow at 0 up to 16% NaCl (w/v), optimally at 3–5% NaCl. The major cellular fatty acids are C18:1ω7c and C19:0cyc and the prevailing quinone is Q-10. The predominant phospholipids are phosphatidylcholine, phosphatidylglycerol, phosphatidyldimethylethanolamine and phosphatidylethanolamine. Assimilate NH4+ by glutamate dehydrogenase and via the glutamate cycle (glutamine synthetase and glutamate synthase). The DNA G + C contents of strains C2T and SK12T are 60.9 and 60.5 mol% (Tm), respectively. 16S rRNA gene sequence similarity between the two new isolates are 99% but below 94% with other members of the Alphaproteobacteria thus indicating that they can be assigned to a novel genus Methyloligella. Rather low level of DNA–DNA relatedness (53%) between the strains C2T and SK12T indicates that they represent two separate species of the new genus, for which the names Methyloligella halotolerans gen. nov., sp. nov. and Methyloligella solikamskensis sp. nov. are proposed. The type strain of Methyloligella halotolerans is C2T (=VKM B-2706T = CCUG 61687T = DSM 25045T) and the type strain of Methyloligella solikamskensis is SK12T (=VKM B-2707T = CCUG 61697T = DSM 25212T). 相似文献
20.
Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5 fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6 pg ml− 1 to a high of 72,962 pg ml− 1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise. 相似文献