Genome size estimation of liver fluke Opisthorchis viverrini by real-time polymerase chain reaction based method

The human liver fluke, Opisthorchis viverrini, has been categorized as a class one carcinogenic organism according to its strong association with cholangiocarcinoma, bile duct cancer which has high incidence in the northeast of Thailand. The lack of genome database of this parasite limited the studies aimed to understand the basic molecular biology of this carcinogenic liver fluke. The determination of the genome size is an initial step prior to the full genome sequencing. In this study, we applied an absolute quantitative real-time polymerase chain reaction for this aspect. Our results indicated the genome size of O. viverrini is 75.95 Mb or C value 0.083. The information of O. viverrini genome size is useful for estimation of sequence coverage and the cost of the parasite's whole genome sequencing using next-generation sequencing technologies.     

Effects of selected insecticides on Cotesia plutellae, endoparasitoid of Plutella xylostella

Effects of field dosages ofselected insecticides to Cotesiaplutellae (Kurdjumov) (Hymenoptera: Braconidae), a larval endoparasitoidof Plutella xylostella L. (Lepidoptera: Plutellidae), wereinvestigated under laboratory conditions.Emergence of adult C. plutellae frominsecticide-treated pupae was not significantlydifferent from the control treatment. Contacttoxicity to C. plutellae adults variedgreatly among the insecticides in a paperresidue contact bioassay. Threeazadirachtin-based insecticides, Agroneem(4.8 mg a.i.liter^–1), Neemix (20 mga.i.liter^–1) and Ecozin (20 mgai.liter^–1) caused 11.1, 16.7 and 5.6%adult mortality, respectively. Of fourcommercial Bacillus thuringiensis (Bt)insecticides examined (all at 1.2 mga.i.liter^–1), Crymax and Xentari had noeffect on adult parasitoids, whereas Mattchcaused 5.6% mortality, and Dipel caused 11.1%mortality. Indoxacarb (53 mg a.i.liter^–1),-cyhalothrin (28 mg a.i.liter^–1) andspinosad (53 mg a.i.liter^–1) caused 100,88.5 and 50% adult mortalities, respectively.Low adult mortality (0–5.6%) was recorded fromingestion of azadirachtin-based, Btinsecticides and indoxacarb, compared with100% adult mortality in treatments of spinosador -cyhalothrin. Compared with the watercontrol, ingestion of azadirachtin-basedinsecticides significantly reduced parasitismby 50–57%, and Bt insecticides by 8–25%.However, ingestion of these insecticides didnot affect longevity of male and femaleparasitoid adults with one exception; femalelongevity was significantly reduced in theindoxacarb treatment. Insecticide residuescaused considerable mortality of C.plutellae adults, 39 and 44% mortality causedby 10 d old indoxacarb and -cyhalothrin,respectively, and 24 and 0% mortality causedby 7 and 10 d old residues of spinosad,respectively.     

Sex allocation and clutch size in the gregarious larval endoparasitoid wasp, Cotesia glomerata

The ability of the gregarious larval endoparasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) to adjust progeny sex ratio and clutch size was investigated. The sex ratios (proportion of males) of field clusters were diverse, but many (70%) were female-biased. Nearly 10% yielded males only, suggesting a low percentage of unmated females in the field. In over half of the clusters containing females, the sex ratio was below 0.3. Superparasitism was common in the field, and females were believed to increase progeny sex ratio when attacking previously-parasitized hosts. However, in a single oviposition bout, sex allocation was not precisely controlled both in the field and laboratory. In the laboratory, the number of eggs laid in a day tended to decrease with increasing female age. For females which were offered two hosts per day and for those offered three hosts per day, this value became nearly the same several days after the start of oviposition. The total number of hosts which a female could parasitize during her lifetime was often less than 40. Some of the old females which attacked more than 40 hosts produced male-biased clutches; this was due to sperm depletion, because sperm remained viable throughout a female's lifetime. The amount of sperm used in a single oviposition bout seemed fixed and was not dependent on the number of eggs laid. Females with much oviposition experience did not produce new eggs to compensate for deposited eggs, and the efficiency of egg use (deposited eggs/total eggs) was more than 80%.     

Exogenous JH and ecdysteroid applications alter initiation of polydnaviral replication in an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera)

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.     

实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础,本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列,Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani)AG1-IA融合群菌株GD118为对照,采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小,研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。     

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction.

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell.     

New buffers to improve the quantitative real-time polymerase chain reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

Estimating mitochondrial DNA content of chinook salmon spermatozoa using quantitative real-time polymerase chain reaction

Animal mitochondrial DNA (mtDNA) is predominantly inherited maternally. Various mechanisms to avoid the transmission of paternal mtDNA to offspring have been proposed, including the dilution of paternal mtDNA by maternal mtDNA in the zygote. The effectiveness of dilution as a barrier will be determined by the number of mtDNA molecules contributed by each parental gamete, and is expected to be highly variable among different taxa due to interspecific differences in mating systems and gamete investment. Estimates of this ratio are currently limited to few mammalian species, and data from other taxa are therefore needed to better understand the mechanisms of mitochondrial inheritance. The present study estimates mtDNA content in salmon sperm, the first nonmammalian vertebrate to be examined. Although highly divergent, it appears that the mtDNA content may be conserved within vertebrate taxa, indicating that the reduction of mtDNA is a key factor of spermatogenesis to ensure mitochondrial functionality on the one hand, and to avoid paternal leakage at a significant or detectable level on the other hand. We employ quantitative real-time PCR (Q-PCR) and demonstrate the accuracy and high reproducibility of our experiments. Furthermore, we compare and evaluate two standard approaches used for the quantification of genes, Q-PCR and blotting methods, in regard to their utility in the accurate quantification of mitochondrial genes.     

Rapid titer determination of baculovirus by quantitative real-time polymerase chain reaction

Titer determination is a prerequisite for the study of viruses. However, the current available methods are tedious and time-consuming. To improve the efficiency of titer determination, we have developed a rapid and simple method for the routine detection of baculovirus titers using a quantitative real-time PCR. This method is based on the amplification of approximately 150-bp fragments located in the coding regions of selected genes. The PCR was found to be quantitative in a range of 10(3) to 10(9) virus particles per 200 microL of supernatant, and the results were closely correlated with titers detected from 50% tissue culture infectious doses (TCID(50)) of baculovirus. This quantitative real-time PCR requires only 30 min to perform, and the entire titer determination can be accomplished within 1 h without the need for cell seeding or further virus dilution and infection. Because this technology is easy to operate, generates data with high precision, and most importantly is very quick, it will certainly be broadly applied for titer determination of baculoviruses in the future.     

The real-time polymerase chain reaction

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.     

Rapid identification of the north-western Pacific billfish species using quantitative real-time polymerase chain reaction techniques

Billfishes are important fishery resources traded and consumed worldwide. As morphological traits are usually removed during processing, molecular methods are applied to identify billfish products. In this study, the approaches of quantitative real-time PCR were developed to identify the six billfish species (Istiompax indica, Istiophorus platypterus, Kajikia audax, Makaira nigricans, Tetrapturus angustirostris and Xiphias gladius) widely distributed in the north-western Pacific Ocean. The developed singleplex systems showed high fidelities to each of the six species via either examining the ΔCt values or melting curve patterns. For samples containing multiple species, individual species are identifiable by a quantitative real-time PCR assay that includes all the singleplex systems. A multiplex system was also developed to identify unknown samples composed of a single species. The methods developed in this study provide a fast and high-throughput manner to identify the north-western Pacific billfish species when morphological traits are unavailable, such as in processed products.     

Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.     

Real-time quantitative polymerase chain reaction

In Charcot-Marie-Tooth type 1A disease (CMTIA), heterozygosity for the peripheral myelin protein 22 (PMP22) duplication increases the gene dose from two to three, whereas, in hereditary neuropathy with liability to pressure palsies (HNPP), heterozygosity for the PMP22 deletion reduces the gene dose from two to one. Thirty-eight Norwegian patients with CMT1, 4 patients with HNPP, 15 asymptomatic family members, and 45 normal controls were studied using the ABI 7700 sequence detection system and the TaqMan method of real-time quantitative polymerase chain reaction (PCR). Using a comparative threshold cycle (Ct) method and albumin as reference gene, the gene copy number by PMP22 gene duplication or deletion on chromosome 17p11.2-12 was quantified. The PMP22 duplication ratio ranged from 1.50 to 2.21, the PMP22 deletion ratio ranged from 0.44 to 0.55, and the PMP22 ratio in normals ranged from 0.82 to 1.27. All samples were run in triplicate, with a mean standard deviation of 0.07 (range 0.01-0.17). Thirty-four of thirty-eight CMT1 patients (89.6%) had the PMP22 duplication and the four HNPP patients had the PMP22 deletion. This was not found in any of the asymptomatic family members or the controls. Real-time quantitative PCR is a sensitive, specific, and reproducible method for diagnosing PMP22 duplication and deletion. The method is fast, allowing 13 patients to be diagnosed in 2 h. It involves no radioisotopes and requires no post-PCR handling. In our opinion, real-time quantitative PCR is the first method of choice in diagnosing PMP22 duplication and deletion.     

Detection of infectious dengue virus by selective real-time quantitative polymerase chain reaction

Dear Editor,The dengue virus(DENV)is a single-stranded positivesense RNA virus that belongs to the family Flaviviridae(Gubler,2002),and has four serotypes,DENV1–DENV4,which are transmitted via Aedes aegypti and Aedes albopictus(Rodriguez-Roche and Gould,2013).It has been reported that more than 50 million dengue infections occur each year(Guzman et al.,2010),and a serious outbreak occurred in the Southern Provinces of