Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.     

Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCR

Current methodology in real-time Polymerase chain reaction (PCR) analysis performs well provided PCR efficiency remains constant over reactions. Yet, small changes in efficiency can lead to large quantification errors. Particularly in biological samples, the possible presence of inhibitors forms a challenge. We present a new approach to single reaction efficiency calculation, called Full Process Kinetics-PCR (FPK-PCR). It combines a kinetically more realistic model with flexible adaptation to the full range of data. By reconstructing the entire chain of cycle efficiencies, rather than restricting the focus on a 'window of application', one extracts additional information and loses a level of arbitrariness. The maximal efficiency estimates returned by the model are comparable in accuracy and precision to both the golden standard of serial dilution and other single reaction efficiency methods. The cycle-to-cycle changes in efficiency, as described by the FPK-PCR procedure, stay considerably closer to the data than those from other S-shaped models. The assessment of individual cycle efficiencies returns more information than other single efficiency methods. It allows in-depth interpretation of real-time PCR data and reconstruction of the fluorescence data, providing quality control. Finally, by implementing a global efficiency model, reproducibility is improved as the selection of a window of application is avoided.     

Validation of real-time RT-PCR assays for mRNA quantification in baboons

Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.     

Analyzing ligation mixtures using a PCR based method

We have developed a simple and effective method (Lig-PCR) for monitoring ligation reactions using PCR and primers that are common to many cloning vectors. Ligation mixtures can directly be used as templates and the results can be analyzed by conventional gel electrophoresis. The PCR products are representative of the recombinant molecules created during ligation and the corresponding transformants. Orientation of inserts can also be determined using an internal primer. The usefulness of this method has been demonstrated using ligation mixtures of two cDNA’s derived from the salivary glands of Aedes aegypti mosquitoes. The method described here is sensitive and easy to perform compared to currently available methods.     

Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.     

Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control

We have developed a quantitative PCR assay (LightCycler* using the pair of primers JW58 and JW59 for the detection of the 35-fold repeated B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106( )parasites in one ml of amniotic fluid (1 to 105( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.     

通用PCR法在痰标本真菌检测中的临床应用价值分析

目的:探讨通用PCR法在痰标本真菌检测中的临床应用价值。方法:选择2015年9月至2017年9月本院收治的免疫力低下患者178例作为研究资料,各收集痰标本2份,其中一份用于PCR扩增,另一份经培养后若观察到真菌或可疑菌落,则提取后在此进行PCR扩增测序。比较3种方法对真菌的检出情况、真菌阳性率,与组织病理学结果进行比较,分析3种方法的诊断效能。结果:178份痰标本经平板培养可观察到107份真菌生长,其余71份未见真菌生长。挑取真菌及可疑菌落行PCR结果显示阳性、阴性分别115、63份。对痰标本直接PCR结果显示,阳性、阴性分别124、54份。PCR扩增产物测序结果显示大多是白色念珠菌感染,仅3份是曲霉菌感染。内对照扩增验证结果显示有1份阴性标本存在扩增抑制现象。3种方法的真菌阳性率:直接PCR法痰培养后PCR法痰培养,但组间无显著差异(P0.05)。3种方法诊断效能总体趋势:直接PCR法痰培养后PCR法痰培养,其中直接PCR法与痰培养后PCR法的特异度、准确度均显著高于痰培养法(P0.05),直接PCR法的敏感度显著高于痰培养法(P0.05)。结论:通用PCR法在痰标本真菌检测中的临床应用价值较高,直接PCR具有操作简单、快速等优势。     

CUT&Tag产物回收和建库方法的优化

新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质免疫共沉淀测序(chromatin immunoprecipitation sequencing,ChIP-seq)的实验过程。CUT&Tag中转座酶完成标签化后需要DNA回收或其他后处理才能进行建库PCR,不同的回收方法对CUT&Tag结果有着显著的影响。通过建立生物素化转座体-链霉亲和素磁珠体系(streptavidin beads recovery CUT&Tag,srCUT&Tag),可以快速便捷地完成CUT&Tag的产物回收。本文在K562细胞中展开H3K4me3、RNA聚合酶Ⅱ(RNA polymeraseⅡ,RNAPⅡ)、转录因子CTCF和HMGA1的CUT&Tag实验,并利用现有的乙醇沉淀、片段分选(solid-phase reversible immobilization,SPRI)磁珠回收和直接PCR法,以及本研究建立的srCUT&Tag方法对产物进行回收。结果表明,从整体上看,SPRI磁珠回收和srCUT&Tag方法着较高的回收效率,而乙醇沉淀法则回收效率低下。在全部4种CUT&Tag产物回收过程中,SPRI磁珠回收均会损失大部分小于150 bp的产物片段。在CTCF和HMGA1 CUT&Tag产物的回收中,直接PCR法则损失了大部分大于300 bp的片段并与其他回收方法的结果有较大的差别。因此,srCUT&Tag能够比其他三种回收方法提供更多更完整的测序信息。综上所述,新建立srCUT&Tag回收方法相比现有的CUT&Tag产物回收方法能提高建库效率并得到更好的数据质量,为表观遗传学研究提供了更好的技术选择。     

Site-specific forced misincorporation mutagenesis using modified T7 DNA polymerase

A new method for forced misincorporation site-specific mutagenesis is described. The method uses an exonuclease-deficient modified version of T7 DNA polymerase in the presence of one dNTP to force a misincorporation. Analysis by PAGE is used to monitor the efficiency of such misincorporation reactions. Brief extension of the terminally mismatched primer/template using the same enzyme in the presence of all four dNTPs is followed by chase/ligation using unmodified T7 DNA polymerase and T4 DNA ligase to give heteroduplex DNA. We have applied the method to mutagenesis of the Lac Z region of M13 and found that, using strand selection, efficiencies of mutagenesis at one site are greater than 50%. When the mutating dNTP is complementary to a neighbouring homopolymeric tract on the template, multiple mutation is observed and efficiences are lower. The method is more general than internal mismatch mutagenesis and, because of its rapidity, is more expedient than existing methods of forced misincorporation mutagenesis.     

Work efficiency: a new criterion for comprehensive comparison and evaluation of statistical methods in large-scale identification of differentially expressed genes

Receiver operating characteristic (ROC) has been widely used to evaluate statistical methods, but a fatal problem is that ROC cannot evaluate estimation of the false discovery rate (FDR) of a statistical method and hence the area under of curve as a criterion cannot tell us if a statistical method is conservative. To address this issue, we propose an alternative criterion, work efficiency. Work efficiency is defined as the product of the power and degree of conservativeness of a statistical method. We conducted large-scale simulation comparisons among the optimizing discovery procedure (ODP), the Bonferroni (B-) procedure, Local FDR (Localfdr), ranking analysis of the F-statistics (RAF), the Benjamini-Hochberg (BH-) procedure, and significance analysis of microarray data (SAM). The results show that ODP, SAM, and the B-procedure perform with low efficiencies while the BH-procedure, RAF, and Localfdr work with higher efficiency. ODP and SAM have the same ROC curves but their efficiencies are significantly different.     

Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.     

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct.MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations.ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R² >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations).ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.     

Why is the polymerase chain reaction resistant to in vitro evolution?

A variety of methods have been developed to amplify DNA and RNA. These methods vary in their susceptibility to evolve new molecular species differing from the starting template. PCR is exceptionally resistant to in vitro evolution, whereas methods such as Q replicase and 3SR are much less robust. This paper develops some simple mathematical models which suggest that PCR is resistant to in vitro evolution because the reaction controls replication in discrete cycles: fast replication is of little advantage during PCR because the reaction limits fast replicators as well as slow ones to a single copy per cycle. In contrast, continuous (isothermal) reactions, as in the Q replicase reaction, favor fast replicators. The advantage of fast replication is compounded in continuous reactions, because a fast replicator can complete many generations of replication during the time it takes a slow replicator to complete one generation. These models suggest that continuous amplication protocols will never achieve the robustness against in vitro evolution observed with PCR.Correspondence to: J.J. Bull     

Rapid one-step recombinational cloning

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning.     

Kinetic Outlier Detection (KOD) in real-time PCR

Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3–1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.     

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.     

Histochemical classification based upon reaction types and its application to carbohydrate histochemistry

Summary A method of classification is presented, which divides histochemical visualization reactions into categories based on general reaction types. This scheme is dependent upon the reaction between two elements, the substrate and the probe. The substrate represents a tissue component(s) with one or more reactive groups that can combine directly with the probe. Alternatively, the substrate reactive groups are chemically modified (activation) with a suitable reagent before reaction with the probe. Probes are of three types: those that yield a coloured product, those that result in a colourless product, and those that produce a coloured product only after a further reaction.Methods used in carbohydrate histochemistry are divided into one, two and three probe reactions. Two probe reactions are further subdivided into sequences involving one or two coloured products (one and two dye sequences); three probe reactions into sequences involving one, two or three coloured products (one, two and three dye sequences). This classification permits the rationalization and organization of methods, and provides a framework for the examination of existing methods and the development of new ones.