Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting

Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay (‘GenoType MTBC’ from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay (‘MTB LAMP’) showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay (‘Muniv LAMP’) showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.     

Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC ≥ 100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC < 25 mg/L) MRSA (100% negative). They were performed under the same reaction conditions within 60 min at 63 °C. The combined LAMP assays will be useful for rapid identification of S. aureus isolates and determination of their antibiotic and antiseptic resistance patterns with regard to methicillin and organic cationic substrates.     

Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25 min at 63 °C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (T_m) of ∼89 °C. The assay analytical sensitivity is ∼0.1 tryps/ml while that of classical PCR test targeting the same gene is ∼10 tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves,straw sizes,and thawing rates

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (−10 °C/min) or a fast (−40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8 s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P < 0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P > 0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P < 0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P > 0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples thawed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P < 0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.     

Diapause induction, maintenance and termination in the rice stem borer Chilo suppressalis (Walker)

The rice stem borer, Chilo suppressalis, enters facultative diapause as fully grown larvae in response to short-day conditions during the autumn. Our results showed that the critical night length for diapause induction in C. suppressalis was between 10 h 22 min and 10 h 45 min at 22, 25 and 28 °C, 11 h 18 min at 31 °C, and between 10 h 5 min and 10 h 20 min under field conditions (average temperature ranged from 27.2 to 30.7 °C). The diapause incidence declined in ultra-long nights (18-22 h scotophases) and DD, and increased in ultra-short nights (2-6 h scotophases) and LL. Moreover, we found that the third instar was the stage most sensitive to the photoperiod, and night length played an essential role in the initiation of diapause. Night-interruption experiments with a 1-h light pulse at LD 12:12 (light 12:dark 12) exhibited two troughs of diapause inhibition, with one occurring in early scotophase and the other in late scotophase. Field observations for six years showed that most larvae entered winter diapause in August in response to declining day lengths, despite the high temperatures prevailing during August. By periodically transferring the field-collected overwintering larvae to different photoperiods and temperatures, the results showed that photoperiod had a significant influence on diapause development during the early phase of diapause, while high temperature significantly accelerated the termination of larval diapause.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Intraspecific tolerance of Metarhizium anisopliae conidia to the upper thermal limits of summer with a description of a quantitative assay system

Conidial tolerance to the upper thermal limits of summer is important for fungal biocontrol agents, whose conidia are formulated into mycoinsecticides for field application. To develop an efficient assay system, aerial conidia of eight Metarhizium anisopliae, four M. anisopliae var. anisopliae, and six M. anisopliae var. acridum isolates with different host and geographic origins were wet-stressed for ≤180 min at 48 °C or incubated for 14 d colony growths at 10-35 °C. The survival ratios (relative to unstressed conidia) of each isolate, examined at 15-min intervals, fit a logistic equation (r² ≥ 0.975), yielding median lethal times (LT₅₀s) of 14.3-150.3 min for the 18 isolates stressed at 48 °C. Seven grasshopper isolates from Africa had a mean LT₅₀ of 110 (73-150) min, but could not grow at 10 or 15 °C. The mean LT₅₀ of five non-grasshopper isolates capable of growing at 10-35 °C was 16 (10-26) min only. Three isolates with typically low (type I), medium (type II), and high (type III) levels of tolerance to 48 °C were further assayed for ≤4-d tolerance of their conidia to the wet stress at 38, 40, 42, or 45 °C. The resultant LT₅₀s decreased to 20, 53 and 167 min at 48 °C from 507, 1612, and 8256 min at 38 °C for types I, II and III, respectively. For the distinguished types, the logarithms of the LT₅₀s were significantly correlated to the temperatures of 38-48 °C with an inverse linearity (r² ≥ 0.88). The method developed to assay quantitatively fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.     

Identification of zoonotic Giardia genotypes in fish

Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in fish. The present study investigated the prevalence of Giardia in cultured fingerlings (n = 227), wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of fish in Western Australia by PCR amplification at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in fish hosts. The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E and Giardia microti were detected. The identification of zoonotic species of Giardia highlights the public health importance of investigating parasites within fish host species.     

Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery

Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1-20 mg L⁻¹ of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g⁻¹) than S. subsalsa (1.32 mg g⁻¹) at pH 6 within 3 h of exposure to 20 mg L⁻¹ Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 μM) did not influence Pb accumulation whereas 100 μM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L⁻¹ Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption-desorption is possible.     

Release of monomeric sugars from Miscanthus sinensis by microwave-assisted ammonia and phosphoric acid treatments

Microwave-assisted ammonium hydroxide (NH₄OH) followed by phosphoric acid (H₃PO₄) treatments were used to release monomeric sugars from Miscanthus sinensis grown in Cha-Chueng-Sao province, Thailand. Treatment with 1.0% (w/v) NH₄OH, 15:1 liquid-to-solid ratio (LSR) at 120 °C temperature for 15 min liberated 2.9 g of monomeric sugars per 100 g of dried biomass, whereas the corresponding yield for a treatment with 1.78% v/v H₃PO₄, 15:1 LSR at 140 °C for 30 min was 62.3 g/100 g. The two-stage pretreatment, treatment with NH₄OH at 120 °C temperature for 15 min followed by treatment with H₃PO₄ at 140 °C for 30 min, impressively provided the highest total monomeric sugar yield of 71.6 g/100 g dried biomass.     

Synthesis and evaluation of anti-tubercular activity of new dithiocarbamate sugar derivatives

The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-β-d-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1 by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25-12.5 μg/ml. The antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Importantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bactericidal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in contrast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent compound against MTB, particularly promising to overcome drug resistance.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

New microsporidia parasitizing bark lice (Insecta: Psocoptera)

Two species of bark lice, Xanthocaecilius sommermanae Mockford and Polypsocus corruptus Hagen, collected in a canopy Malaise trap placed in Great Smoky Mountains National Park as part of a survey of the park’s fauna, were found to be infected with microsporidia. Diagnosis was originally based on light microscopy, and was confirmed by PCR amplification and electron microscopy. This is the first record of microsporidia infection in the insect order Psocoptera. Four morphological spore types corresponded to four original SSUrDNA sequences (Genbank accession no. FJ865221-24), suggesting infection with four microsporidia species. Two of those species were examined by electron microscopy. We describe here one new genus and two new species based on morphological and sequence data: Antonospora psocopterae sp. n. with elongated diplokaryotic spores, 4.4 ± 0.05 × 1.9 ± 0.03 μm and Mockfordia xanthocaeciliae gen. n. sp. n. with ovocylindrical monokaryotic spores, 2.5 ± 0.10 × 1.4 ± 0.02 μm. A. psocopterae displayed high sequence (95%) and structural similarity with Antonospora scoticae, fell within a well supported dichotomy with A. scoticae inside the Antonospora-Paranosema clade in phylogenetic analyses by NJ, PS and ML. M. xanthocaeciliae did not exhibit much sequence or structural similarity with any of known microsporidia species, except Encephalitozoon spp. M. xanthocaeciliae fell within one clade with Encephalitozoon spp. in phylogenies and shared with encephalitozoons structural resemblance and about 80% of SSUrDNA sequence identity. The other two species were not described and provisionally were placed to the collective genus Microsporidium as Microsporidium sp. 1 and Microsporidium sp. 4 from bark lice because of insufficient morphological data. The finding that samples fixed and stored for months in propylene glycol (“antifreeze”) are good enough for DNA sequence analysis and can be used for morphological analyses (if no better fixation alternatives are available), is promising for future surveys for microsporidia.     

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.     

Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device

We present a thermal gradient convective polymerase chain reaction (PCR) for parallel DNA amplification with different annealing temperatures. The thermal gradient for microfluidic gradient PCR is produced by an innovative fin design whose formation principle is given. Without the need for a pump, the buoyancy forces continuously circulate reagents in a closed loop through different thermal zones, which brings self-actuated convective-flow PCR. In our prototype, we measured a temperature difference of about 45 °C along the gradient direction on the copper flake (45 × 40 × 4 mm). When the temperature of the hot zone is 90-97 °C and the temperature of the cold zone is 60-70 °C, the convection triggered two-temperature amplification of 112-bp fragment of Escherichia coli DNA. The time for amplification is less than 45 min. Interestingly, parallel DNA amplification with different annealing temperatures ranging from 60 to 70 °C was performed by this method. The PCR thermocycler demonstrated herein can be further scaled down and the loop length can be further reduced, and therefore the PCR times can be further reduced. These devices are suited as a platform for a new generation of low-power, portable DNA analysis systems.     

Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification

In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by droplet-vitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L⁻¹ sucrose for 2 days, osmoprotected in loading solution for 20-40 min at room temperature and then soaked in PVS2 solution for 90-120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method.     

Construction and characterization of a copy number-inducible fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was > 34 kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5 kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5 hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.