Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

This meta-analysis was designed to assess the effect of the addition of a bead-beating (BB) step during DNA extraction to effectively isolate Trichuris trichiura DNA from stool samples for quantitative polymerase chain reaction (qPCR)-based diagnosis. qPCR-based molecular studies comparing the inclusion of a bead-beating step during the DNA extraction from stool samples with extraction without the step were included in the analysis. Studies using real patient samples in community settings were included. The PubMed database and Google search engine were searched in December 2019. Risk of bias and applicability were assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 checklist. Odds ratios (ORs) for individual studies were combined to estimate the random effects model OR. A total of six independent sub-studies were gathered from two published original articles. The division of the two major studies into six sub-studies was indispensable due to the nature of the study carried out. 128 of the total 192 samples (in all studies) were positive for T. trichiura when BB was used during DNA extraction compared to 108/192 when BB was excluded. The combined OR was 1.66 (95% confidence interval: 1.059 to 2.602). Though only two articles were included in the study, six exclusive individual sub-studies were analyzed. Inherent differences in the background prevalence of helminths in the study population could impact the sensitivity of qPCR. It was found that the inclusion of the BB step during DNA extraction significantly increased the sensitivity of the test. This study was not registered in any database.

Sedimentary ancient DNA (sedaDNA) has been established as a viable biomolecular proxy for tracking taxon presence through time in a local environment, even in the total absence of surviving tissues. SedaDNA is thought to survive through mineral binding, facilitating long-term biomolecular preservation, but also challenging DNA isolation. Two common limitations in sedaDNA extraction are the carryover of other substances that inhibit enzymatic reactions, and the loss of authentic sedaDNA when attempting to reduce inhibitor co-elution. Here, we present a sedaDNA extraction procedure paired with targeted enrichment intended to maximize DNA recovery. Our procedure exhibits a 7.7–19.3x increase in on-target plant and animal sedaDNA compared to a commercial soil extraction kit, and a 1.2–59.9x increase compared to a metabarcoding approach. To illustrate the effectiveness of our cold spin extraction and PalaeoChip capture enrichment approach, we present results for the diachronic presence of plants and animals from Yukon permafrost samples dating to the Pleistocene-Holocene transition, and discuss new potential evidence for the late survival (~9700 years ago) of mammoth (Mammuthus sp.) and horse (Equus sp.) in the Klondike region of Yukon, Canada. This enrichment approach translates to a more taxonomically diverse dataset and improved on-target sequencing.

Grapevine cultivar identification is based mainly on two complementary methodologies: microsatellite (simple sequence repeat (SSR)) DNA analysis and traditional ampelography. Here, we report a direct multiplex PCR approach that allows the simultaneous amplification of 11 SSR loci from crude samples, i.e. bypassing DNA extraction, by using an engineered DNA polymerase improved to tolerate plant PCR inhibitors. Many different plant tissues were successfully amplified: leaf, root, wood, berry flesh and skin, stalk and must, from wine and table grape varieties, and rootstocks. The direct multiplex PCR that we propose is quicker and cheaper than the methodologies used until now, and provides accurate results. Thus, SSR DNA analysis becomes economically more accessible to a larger number of potential users in addition to research institutes.

In order to identify Echinococcus multilocularis DNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol–chloroform extraction and a purification step on a matrix (Prep-A-Gene®). The target sequence for amplification was the E. multilocularis U1 snRNA gene. PCR products were indistinguishable for 32 different E. multilocularis isolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, including E. granulosus. The sensitivity of amplification was monitored by the addition of E. multilocularis DNA or eggs to faeces free of E. multilocularis and was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which no E. multilocularis adult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouring E. multilocularis adult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosus and E. multilocularis), or to DNA variation between different isolates of E. multilocularis. Further work in the field should be initiated in order to confirm these results.

Investigating the spatio-temporal patterns of planktonic larvae is fundamental to studies regarding stock assessment and larval dispersal of commercial and non-commercial, i.e. invasive or rare marine invertebrates. Because of the difficulty involved in morphological identification of marine invertebrate larvae, various molecular methods based on PCR have been used to enhance taxonomic resolution. In previous studies, different methods for the preservation or pretreatment of larvae were applied in each case. However, no comparative studies have been conducted to determine the optimal method for PCR testing for bivalve larvae, and no information is available regarding the selection of an appropriate method.

This study compared the PCR success rate of 6 pretreatment methods for larvae of the Mediterranean blue mussel, which was preserved using different fixatives (70% ethanol, 100% ethanol, 70% acetone and 10% formalin). The results revealed that the success rate of PCR was different for each pretreatment; moreover, the use of ammonium sulphate and Tween 20 buffer with proteinase K digestion was found to be the most effective method. Some pretreatments showed lower success rates for long-fixed larvae than for short-fixed larvae for formalin-fixed larvae; however, the success rate of PCR amplification for ethanol-fixed larvae pretreated by this method did not decrease through 1-year fixation. In addition, this pretreatment showed a high success rate for different fixation periods. These findings suggest that the selection of the pretreatment method is critically important for successfully amplifying larval DNA and that the pretreatment involving the use of ammonium sulphate prior to PCR amplification enables the use of fixatives for preserving bivalve larvae. This method will be utilized in various field studies and molecular genetic studies.

Molecular techniques involving polymerase chain reaction (PCR) and sequencing provide a relatively simple and objective means of identifying microsporidia to species level. We modified previously described methods of DNA extraction and PCR conditions for identification of microsporidia from museum slides, clinical specimens and environmental samples and successfully identified Vittaforma corneae in 11 out of 13 cases of microsporidial infection from used trichrome-stained slides of corneal scrapings from HIV-negative patients with keratoconjunctivitis.

The peach–potato aphid Myzus persicae (Sulzer) is an important vector of plant viruses. A network of suction traps collects aerial samples of this aphid in order to monitor and help predict its spatial distribution and likely impact on virus transmission in crops. A suction trap catch is thought to be a good representation of the total aphid pool. Sensitive molecular markers have been developed that determine the genetic composition of the M. persicae population. In Scotland, UK, these were applied to field collections revealing a limited number of clones. Molecular markers are less successful when applied to specimens that have been preserved in an ethanol-based trap fluid designed to preserve morphology. An assessment of different DNA extraction and PCR techniques is presented and the most efficient are used to analyse M. persicae specimens caught in the Dundee suction trap in 2001, a year when exceptionally high numbers were caught. The results reveal that the majority of the M. persicae caught belonged to two highly insecticide resistant clones. In addition, it was possible to compare the relative frequencies of genotypes caught in the trap with those collected at insecticide treated and untreated field sites in the vicinity. These results indicate that, in addition to suction trap data, the ability to sample field sites provides valuable early warning data which have implications for pest control and virus management strategies.

A modified technique to extract intact DNA from Aedes aegypti and Anopheles darlingi mosquitoes is presented. The samples were treated initially with proteinase K to digest all proteinaceous matter. For both vectors, the optimum temperature for the protease treatment was 55°C and the best pre-incubation time was 4 h. RNA contamination was eliminated with RNAse treatment. The purity of the DNA was high since the A260/A280 ratio averaged >1.80 for all samples and the quantity of DNA extracted was >1.6 times higher than that using the unmodified procedure. The DNA obtained displayed clear random amplified polymorphic DNA (RAPD) profiles, which indicates that it can be used for polymerase chain reaction (PCR)-based techniques.

A simple, rapid and low-cost method for the isolation of high molecular weight DNA from small amounts of cells or tissue is described. The method does not require liquid nitrogen, proteinase K or costly buffers. This method was successfully applied in the isolation and purification of DNA from eggs of Hyalomma excavatum, H. dromedarii, Argas persicus, A. hermanii and Boophilus annulatus, in addition to Escherichia coli, actinomycetes (Nocardia sp.) and leaves of soybean (Glycine max L.). The DNA concentration was calculated from the optical density (OD) value at wavelength 260 nm and the ratios OD260/OD280 and OD260/OD280 determined. The quality and amount of DNA obtained by this method were comparable to those of DNA isolated from equal amounts of material using a commercial genomie DNA purification kit.