Simple Polymerase Chain Reaction-restriction Fragment Research Articles

Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

Guidelines for laboratory diagnosis of factor XIII deficiency

Factor XIII (FXIII) deficiency is an extremely rare hemorrhagic disorder with an approximate worldwide incidence of one per two million. With current tests, diagnosis of this disease can be made more precisely. However, factors such as the number of patients with FXIII deficiency (FXIIID), available diagnostic coagulation tests and the number of molecular studies have affected the diagnosis of FXIIID in different parts of the world. Various laboratory approaches can be used, including screening and diagnosis of the disorder in countries with a relatively high rate of FXIIID and recurrent mutation(s) with a simple polymerase chain reaction-restriction fragment length polymorphism analysis or polymerase chain reaction-sequencing for detection of one or a few specific mutations. In other countries, two different laboratory approaches can be used, depending on available coagulation tests. In less-equipped coagulation laboratories, the clot solubility test remains the only diagnostic test for FXIIID. Even in these countries, at least one referral laboratory should perform FXIII activity and, if possible, confirmation of FXIIID by molecular analysis. In countries with well equipped coagulation laboratories, FXIII activity should be used to screen suspected FXIIID patients; more specific tests such as molecular analysis should be used for confirmation. This study suggests a simple, reliable and flexible algorithm for early diagnosis of FXIIID, and may, with one-time diagnosis of FXIIID, reduce the rate of morbidity and mortality in patients with the disorder.

Identification of a point mutation in the ace1 gene of Therioaphis trifolli maculata and detection of insecticide resistance by a diagnostic PCR-RFLP assay.

Aphids are important agricultural pests worldwide. Their control is largely based on chemical insecticides. One species that shows important invasive abilities and host-plant-related differences is Therioaphis trifolii (Monell) (Hemiptera: Aphididae). T. trifolii maculata, also known as spotted alfalfa aphid (SAA), can be very injurious to alfalfa crops in certain regions, such as in Saudi Arabia for effective control it is essential to diagnose and monitor the resistance mechanisms in the SAA populations. In the present study, we analysed acetylcholinesterase (ace) target site insensitivity mechanisms. A 650 bp length DNA containing the putative acetylcholinesterase (ace1) precursor was obtained and compared with other Hemipteran species. The sequences of many individual aphids collected from alfalfa crops in Saudi Arabia were analysed for the presence of resistance mutations: no resistance mutations were found at the resistance mutation loci 302; however, the presence of a serine-phenylalanine substitution (S431F) was identified in one individual. The S431F substitution, has been shown to confer significant levels of both organophosphate and carbamate resistance in other aphid species, and is now found for the first time in T. trifolii. We subsequently developed a simple polymerase chain reaction-restriction fragment length polymorphism assays for the S431F mutation, using a TaqI restriction site destroyed by the S431F mutation. The novel diagnostic assay may support the implementation of Insecticide Resistance Management strategies, for the control of SAA in alfalfa crops in the Kingdom of Saudi Arabia, and other countries worldwide.

Mitochondrial DNA polymorphisms in Phytophthora infestans: New haplotypes are identified and re-defined by PCR

Polymorphisms of mitochondrial DNA (mt-DNA) are particularly useful for monitoring specific pathogen populations like Phytophthora infestans. Basically type I and II of P. infestans mt-DNA were categorized by means of polymorphism lengths caused by an ~2kb insertion, which can be detected via restriction enzyme digestion. In addition genome sequencing of haplotype Ib has been used as a simple Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR–RFLP) method to indirectly identify type I and II alterations through EcoR I restriction enzyme DNA fragment patterns of the genomic P4 area. However, with the common method, wrong mt-DNA typing occurs due to an EcoR I recognition site mutation in the P4 genomic area. Genome sequencing of the four haplotypes (Ia, Ib, IIa, and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and we indentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRi length polymorphism caused by a 2kb insertion/deletion was utilized to identify mt-DNA types I and II, while another length polymorphism in the HVRii region is caused by a variable number of tandem repeats (n=1, 2, or 3) of a 36bp sized DNA stretch and was further used to determine mt-DNA sub-types, which were described as Rn=1, 2, or 3. Finally, the P. infestans mt-DNA haplotypes were re-defined as IR1 or IIR2 according to PCR derived HVRi and HVRii length polymorphisms. Twenty-three isolates were chosen to verify the feasibility of our new approach for identifying mt-DNA haplotypes and a total of five haplotypes (IR1, IR2, IR3, IIR2 and IIR3) were identified. Additionally, we found that six isolates determined as type I by our method were mistakenly identified as type II by the PCR–RFLP technique. In conclusion, we propose a simple and rapid PCR method for identification of mt-DNA haplotypes based on sequence analyses of the mitochondrial P. infestans genome.

Introducing human population biology through an easy laboratory exercise on mitochondrial DNA

This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of different sizes that can be visualized in agarose gels. The analysis of these fragments can reveal the mitochondrial haplogroup of each student. The results of the exercise can be used to provide additional insights into the genetic variation of human populations.

Applications of PCR-RFLPs for differentiating two freshwater sponges: Ephydatia fluviatilis and Ephydatia mülleri

The two freshwater sponges Ephydatia fluviatilis and Ephydatia mulleri belong to the widespread Spongillidae family. Their morphological tracts are very similar and can be distinguished mainly on the basis of their gemmuloscleres. However, when gemmules are absent it is essential to have an unambiguous species attribution for a population genetic study based on fresh tissues and historical collections. This article reports a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, applied to DNA extracted from both gemmules and fresh tissues in order to discriminate between the two congeneric E. fluviatilis and E. mulleri. Such a biomolecular method is based on the discriminative enzymes’ digestion of each of the three amplified fragments 5.8S-ITS2-28S, D3 domain of the 28S subunit and COI. Two restriction enzymes were tested for a 620–642 bp fragment of 5.8S-ITS2-28S and for a 342 bp fragment of the D3 domain of the 28S, one restriction enzyme was tested for a 681 bp fragment codifying for COI. Obtained digestion patterns were diagnostic for each of the two species, providing a relatively simple, fast and cheap method for species attribution compared to sequencing.

A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the differentiation of cephalopod mollusc families Loliginidae from Ommastrephidae, to avoid substitutions in fishery field

In order to provide a tool for the prevention of commercial frauds in fish products, a simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method has been developed. This method allows to clearly differentiate molluscs belonging to the family Loliginidae from those belonging to the family Ommastrephidae. Cephalopods' 16S r-DNA was amplified with PCR using a “universal” primer pair, and the amplification product was digested with AsnI restriction enzyme. The resulting electrophoretic patterns of families Loliginidae and Ommastrephidae showed characteristic 200 bp band and 600–700 bp band, respectively. With this methodology, the analysed species belonging to the genus Loligo have also been differentiated.

Genetic polymorphism at the beta-tubulin locus among human and animal isolates of Cryptosporidium parvum.

Sequence analysis of a fragment of the beta-tubulin gene was performed on 13 isolates of the parasite Cryptosporidium parvum, eight from humans and five from animals. A total of 12 synonymous substitutions and a deletion of two bases within the intron sequence were found. This genetic variation defined two alleles at the beta-tubulin locus, which can be identified by a simple polymerase chain reaction-restriction fragment length polymorphism assay. A total of 20 isolates were also tested using four available molecular markers. These analyses showed congruently that the C. parvum isolates segregate into two groups, one found exclusively in humans and the other found in both humans and animals. Since no recombinant genotypes were observed, the results are consistent with the hypothesis of a substantially clonal reproduction in this parasite.

Genetic polymorphism at the β-tubulin locus among human and animal isolates of Cryptosporidium parvum

Sequence analysis of a fragment of the β-tubulin gene was performed on 13 isolates of the parasite Cryptosporidium parvum, eight from humans and five from animals. A total of 12 synonymous substitutions and a deletion of two bases within the intron sequence were found. This genetic variation defined two alleles at the β-tubulin locus, which can be identified by a simple polymerase chain reaction-restriction fragment length polymorphism assay. A total of 20 isolates were also tested using four available molecular markers. These analyses showed congruently that the C. parvum isolates segregate into two groups, one found exclusively in humans and the other found in both humans and animals. Since no recombinant genotypes were observed, the results are consistent with the hypothesis of a substantially clonal reproduction in this parasite.

ABO genotyping by polymerase chain reaction-restriction fragment length polymorphism

Abstract Genotyping enables the identification of both maternally and paternally derived alleles. A number of protocols have been described for the genotyping of the ABO blood group system. Generally, these methods have a number of disadvantages including the use of hazardous reagents, being technically demanding, and the excessive use of materials. In this study, a relatively simple polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method is described. Four different amplifications were used that were specific for nucleotides sites 261, 526, 703, and 796 to distinguish the A, B, O1 and O2 alleles. The ABO genotypes of 294 random individuals were determined and were found to completely correlate with the serologic phenotypes. The protocol is applicable for investigations of weak or nonexpression of ABO alleles, paternity determinations, and population analysis.