Amplification Success Rate Research Articles

Cross Species Amplification of Coat Colour Genes in Nutria (Myocastor coypus Mol).

In this study, nutria-specific DNA fragments were amplified with cross-species primers for parts of coat colour genes (MC1R, ASIP, TYRP-1, c-KIT) conserved across a number of species. Twenty-three nutria-specific DNA fragments were generated using cross-species PCR (26.7% amplification success rate). Certain limitations of this approach were encountered, including strong mismatching in the primer binding sites. Sequences of 12 nutria-specific PCR products aligned in the BLASTx option showed homology with known protein sequences. Obtained sequences were submitted to GenBank with accession number KC758969 for MC1R, KF511655 for ASIP, KF511656 for TYPR-1 and KU831489 for c-KIT. The highest homology for all translated queries was obtained with protein sequences belonging to species of the rodent family. Sequences of proteins encoded by genes involved in environmental adaptation (coat colour genes in rodents) were found to be strongly conserved among species.

Identification of robust microsatellite markers for wild pig fecal DNA

ABSTRACTCollection of fecal samples for use in a genetic capture‐mark‐recapture framework has become popular as a noninvasive method of monitoring wildlife populations. A major caveat to this process, however, is that fecal samples often yield low quality DNA that is prone to genotyping errors, potentially leading to biases in population parameter estimation. Therefore, considerable care is required to identify robust genetic markers, especially in hot or humid conditions that may accelerate DNA degradation. We identified microsatellite loci in wild pig (Sus scrofa) fecal samples that were robust and informative within warm, humid ecosystems. To examine how degradation affected genotyping success, we sampled pig feces across 5 days and calculated how the number of quantitative polymerase chain reaction (qPCR) cycles required to reach the fluorescent threshold (Ct) changed over time. We identified 17 microsatellite loci that had high polymorphism and amplification success and low genotyping error rates (0–0.050 per locus). In the degradation experiment, Ct increased over the 5 days, but in the absence of rain, the majority of samples produced accurate genotypes after 5 days (2,211/2,550 genotypes). Based on the high amplification success and low error rates, even after 5 days of exposure to warm, humid conditions, these loci are useful for estimating population parameters in pig fecal samples. © 2016 The Wildlife Society.

DNA Barcoding of Highly Threatened Sacred Plant - Aegle marmelos L.

DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, the matK chloroplast gene was tested for species identification of highly threatened sacred plant Aegle marmelos L., to document their reliable identification, discrimination, similarities and evolutionary trend with their related taxa for future use. DNA isolation from leaf sample of Aegle marmelos was carried out by using a modified Cetyl Trimethyl Ammonium Bromide method (CTAB). Gradient PCR amplification was performed for the isolated DNA using matK gene and the primers, matK472F and matK1248R. Pairwise sequence alignments were made with BLAST and multiple sequence alignments were also made with ClustalW. Based on the sequence alignments, dendrograms were constructed using the software, Mega 5 and Neighbor Joining tree method was used to study the phylogenetic aspects of the species studied and with their related taxa. The success rate of PCR amplification was 90-95% and the success rate of DNA sequence was 95%. The maximum identity of the present study sample was 100% with Aegle marmelos and the similarity of other related species, Severinia buxifolia, Citrus trifoliata and Citrus reticulata was 97% respectively.

Metabarcoding of marine zooplankton: prospects, progress and pitfalls

Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few orthologous DNA regions, called barcodes) is revolutionizing analysis of biodiversity of marine zooplankton assemblages. Metabarcoding relies on high-throughput DNA sequencing (HTS) technologies, which yield millions of DNA sequences in parallel and allow large-scale analysis of environmental samples. Metabarcoding studies of marine zooplankton have used various regions of nuclear small- (18S) and large-subunit (28S) rRNA, which allow accurate classification of novel sequences and reliable amplification with consensus primers, but- due to their relatively conserved nature- may underestimate species diversity in a community. To discriminate species, more variable genes are needed. A limited number of metabarcoding studies have used mitochondrial cytochrome oxidase I (COI), which ensures detection of species-level diversity, but may require group-specific primers and thus result in inconsistent amplification success rates. Reference databases with sequences for accurately-identified species are critically needed to allow taxonomic designation of molecular operational taxonomic units (MOTU) and comparison with previous studies of zooplankton diversity. Potential and promising applications of metabarcoding include rapid detection of impacts of climate change, monitoring and assessment of ecosystem health, calculation of biotic indices, characterization of food webs and detection of introduced, non-indigenous species.

Use of the Mitochondrial Control Region as a Potential DNA Mini-Barcoding Target for the Identification of Canned Tuna Species

In this study, a DNA mini-barcoding methodology was developed for the differentiation of species commonly found in canned tuna. Primers were designed to target a 236-base pair (bp) fragment of the mitochondrial control region (CR) and a 179-bp fragment of the first internal transcribed spacer region (ITS1). Phylogenetic analysis revealed the ability to differentiate 13 tuna species on the basis of the CR mini-barcode, except in a few cases of species introgression. Supplementary use of ITS1 allowed for differentiation of introgressed Atlantic bluefin tuna (Thunnus thynnus) and albacore tuna (Thunnus alalunga), while differentiation of introgressed Atlantic bluefin tuna and Pacific bluefin tuna (Thunnus orientalis) requires a longer stretch of the CR. After primer design, a market sample of 53 commercially canned tuna products was collected for testing. This mini-barcoding system was able to successfully identify species in 23 of the products, including albacore tuna, yellowfin tuna (Thunnus albacares), and skipjack tuna (Katsuwonus pelamis). One instance of mislabeling was detected, in which striped bonito (Sarda orientalis) was identified in a product labeled as tongol tuna (Thunnus tonggol). PCR amplification and sequencing was unsuccessful in a number of products, likely due to factors such as the presence of PCR inhibitors and DNA fragmentation during the canning process. Overall, CR and ITS1 show high potential for use in identification of canned tuna products; however, further optimization of the assay may be necessary in order to improve amplification and sequencing success rates.

Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains

Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types.

Evaluation of a commercial real-time PCR kit for the detection of the Q80K polymorphism in plasma from HCV genotype 1a infected patients

BackgroundScreening of the natural HCV NS3 polymorphism Q80K is required prior to simeprevir administration due to the reduced susceptibility of genotype 1 viruses carrying this amino acid variant. A simple, rapid and robust test for Q80K screening would be advisable in routine diagnostic laboratories. ObjectivesThe aim of this study was to evaluate a commercial NS3 Q80K real-time PCR kit (Q80K Polymorphism Kit, Clonit srl, Milan, Italy). Study designForty-three plasma samples obtained from untreated HCV genotype 1a-infected patients and previously sequenced at a reference laboratory, were sent to two public clinical virology laboratories for blinded Q80K screening with the kit under evaluation. The sample panel included 25 cases with the wild type 80Q, 17 with the mutant 80K and 1 with the mutant 80L. ResultsLaboratory 1 identified 22/25 (88.0%) 80Q and 17/17 (100.0%) 80K cases. Laboratory 2 identified 23/25 (92.0%) 80Q and 16/17 (94.2%) 80K cases. All of the unidentified cases were scored as negative, with no mutant/wild type miscalling. The 80L variant was scored as indeterminate by Laboratory 1 and as negative by Laboratory 2. Overall, sensitivity and specificity for detection of 80K were 97.1% (95% C.I., 82.9–99.8%) and 100.0% (90.2–100.0%), respectively. However, the system did not provide any result for 6/84 cases (7.1% failure rate), not including the 80L variant which is not expected to be detected as stated in the kit package insert. Global inter-laboratory concordance was 93.0%. ConclusionsDespite good specificity, this Q80K detection system needs improvements in amplification success rate and robustness.

植物の法科学的検査に適したPCRキットの比較

Recently, plant DNA has been utilized to discriminate or identify forensic botanical samples. In a forensic examination of a plant, various species and plant parts are considered as evidence. PCR amplification has sometimes failed because the samples contain some PCR inhibitors. In this study, to increase the success rate of PCR amplification of plant materials, four kinds of commercially available PCR kits, which showed resistance to PCR inhibitors, were evaluated by two experiments. One experiment was PCR amplification with the addition of chemicals to PCR solution; the other one was PCR amplification using non-purified DNA extracted from plants. The concentrations of chemicals, which inhibited PCR amplification, were different between four kits. Humic acid showed a high inhibitory power, and the concentration of it inhibiting PCR amplification was smaller than the other chemicals by one or two orders of magnitude. PCR amplification from non-purified DNA was succeeded by using a PCR kit, but there was no kit that enabled us to amplify all plant samples. Two kits could amplify more plant samples than the other kits and previous methods. The results indicate that these two PCR kits, which are designed for PCR of plant-derived DNA, are more suitable for forensic examinations of plants.

From the desert it came: evolution of the Australian paper daisy genus Leucochrysum (Asteraceae, Gnaphalieae)

Present patterns of diversity in the Australian flora have been shaped by increasing seasonality since the Eocene, and by pronounced aridification in the past 3 million years. Arid-zone plants are commonly hypothesised to be the products of radiations of ancestrally temperate or coastal lineages, as in the case of the everlasting paper daisy tribe Gnaphalieae (Asteraceae). However, these inferences are often based on higher-level phylogenies, whereas evolutionary processes in the Australian Gnaphalieae have rarely been studied at the species level. Here, we reconstructed the phylogeny and biogeographic history of the small, but ecologically diverse, paper daisy genus Leucochrysum, to examine recent habitat shifts and character changes, at the same time exploring the feasibility of using amplicon sequencing of low-copy nuclear gene regions to resolve phylogenetic relationships in Australian Gnaphalieae. On the balance of evidence, outgroup comparison and ancestral-area reconstruction support an ancestral range in the arid zone with subsequent diversification towards the south-east, demonstrating a complex evolutionary history with a re-colonisation of temperate areas. Low amplification success rates suggest that methods other than amplicon sequencing of currently available primers will be more promising for molecular phylogenetic work at a larger scale.

Identification and persistence of Pinus pollen DNA on cotton fabrics: A forensic application

Advances in plant genomics have had an impact on the field of forensic botany. However, the use of pollen DNA profiling in forensic investigations has yet to be applied. Five volunteers wore a jacket with Pinus echinata pollen-containing cotton swatches for a 14-day period. Pollen decay was evaluated at days 0, 3, 6, 9 and 14 by microscopy. Pollen grains were then transferred to slides using a portable forensic vacuum handle. Ten single grains per swatch were isolated for DNA analysis. DNA was extracted using a high throughput extraction method. A nine-locus short tandem repeat (STR) multiplex system, including previously published primers from Pinus taeda, was developed. DNA was amplified by PCR using fluorescent dyes and analyzed by capillary electrophoresis. Pollen counts from cotton swatches in a 14-day period exhibited an exponential decay from 100% to 17%. The success rate of PCR amplification was 81.2%. Complete and partial STR profiles were generated from 250 pollen grains analyzed (44% and 37%, respectively). Due to the limited amount of DNA, drop-in events were observed (1.87%). However, the rate of contamination with pollen from other pine individuals originating from environmental sources was 4.4%. In conclusion, this study has shown that pollen can be a stable source of forensic DNA evidence, as a proof-of-principle, and that may persist on cotton clothing for at least 14days of wear. This method can be applied in forensic cases where pollen grains larger than 10μm (e.g., from herbs or trees) may be transferred to clothing (worn by suspect or victim) by primary contact.

Noninvasive and nondestructive sampling for avian microsatellite genotyping: a case study on the vulnerable Chinese Egret (Egretta eulophotes)

Noninvasive and nondestructive DNA sampling techniques are becoming more important in genetic studies because they can provide genetic material from wild animals with less or even without disturbance, which is particularly useful for the study of endangered species, i.e., birds. However, nondestructively and noninvasively sampled DNA may, in some cases, be inadequate in the amount and quality of the material collected, which can lead to low amplification success rates and high genotyping errors. In this study, noninvasive (eggshell swab, shed feather and feces), nondestructive (plucked feather and buccal swab) and invasive (blood) DNA samples were collected from the vulnerable Chinese Egret (Egretta eulophotes). DNA concentrations, PCR amplification success and microsatellite genotyping errors of different sample types were evaluated and compared to determine whether noninvasive and nondestructive samples performed as well as invasive samples in our experimental procedures. A total of 159 samples were collected in the field. Among the different sample types, the highest DNA concentrations (154.0–385.5 ng/μL) were obtained from blood. Those extracted from fecal samples were the lowest, ranging from 1.25 to 27.5 ng/μL. Almost all of the DNA samples, i.e., 95.59 %, were successfully amplified for mtDNA (n = 152) and 92.76 % of mtDNA samples were successfully genotyped for at least five of the nine microsatellite loci tested (n = 141). Blood samples and buccal swabs produced reliable genotypes with no genotyping errors, but in feces, allelic dropouts and false alleles occurred in all nine loci, with error rates ranging from 6.67 to 38.10 % for the dropouts and from 6.06 to 15.15 % for the false alleles. These results indicate that both nondestructive and noninvasive samplings are suitable for avian microsatellite genotyping, save for fecal DNA. However, we should remain cautious of the appearance of genotyping errors, especially when using noninvasive material.

ConGenR: rapid determination of consensus genotypes and estimates of genotyping errors from replicated genetic samples

ConGenR (available at http://www.uidaho.edu/cnr/research-outreach/facilities/leecg/publications-and-software ) is an R based conservation genetics script that facilitates rapid determination of consensus genotypes from replicated samples, determines overall (successful amplifications/amplification attempted) and individual sample level (proportion of samples with successful amplifications at n loci) amplification success rates, and quantifies genotyping error rates. ConGenR is intended for use with codominant, multilocus microsatellite data generated primarily through noninvasive genetic sampling and processed with a multi-tubes approach. ConGenR handles input that can be easily exported from GENEMAPPER, a program commonly used to score allele sizes. Amplification success and genotyping error rates can be evaluated by sample class (i.e., any identifiable and meaningful subdivision of samples; e.g., sex, season, region, or sample condition), offering insights into processes driving amplification success and genotyping error rates. Additionally, amplification success and genotyping error rates are calculated by locus, expediting the identification of problematic loci during pilot studies.

SNPforID 52-plex in casework samples: “Cracking” bones and other difficult samples

Casework samples can present difficulties to forensic scientists in criminal and identification investigations. Some challenging samples like bones, teeth and crime scene samples, often contain low DNA quantity which can even be degraded. In these cases, obtained STR profiles are many times incomplete or even null. This is partly due to relative bigger size of commonly used STRs in forensic analysis. In order to bypass this problem, other strategies of analysis have been developed in the past based on mini-STRs and biallelic markers, such as Indels and SNPs. Although each marker type has its advantages and disadvantages, SNPs benefit from the fact of having smaller amplification products and its analysis can be realized analyzing simultaneously a great number of loci using large multiplexes. One of such multiplex is SNPforID 52-plex which analyzes 52 loci, providing good results, as reported by some authors. Taking this in consideration, we compared the amplification success of 53 real casework samples from our casuistic consisting of bones, teeth and other samples using the 52-plex and the Identifier® Plus kit. Mean amplification success rate by loci was of 73% and 43% respectively and 16 out of 36 samples in which STR profiles were not obtained or in which these were poor, generated complete or almost complete SNP profiles. We conclude that the 52-plex can be a valuable tool in the analysis of different types of challenging forensic samples when STRs fail to provide necessary genetic information for identification.

Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study.

The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae). For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium.

A novel method for the identification of seahorses (genus Hippocampus) using cross-species amplifiable microsatellites

Seahorses (genus Hippocampus) are flagship species for marine conservation and evolutionary studies. Wild seahorse populations have declined sharply because of overfishing or other disrupting activities, which are attracting increasing global concern for their threatened status. For study of their population status, the identification of young seahorses by morphological examination has been controversial and remains a difficult task due to overlapping of meristic and morphological characters. Here we reported a simple and sensitive method to identify seahorse species from China based on cross-species microsatellites. The cross-species amplification success rate varied from 40.0% to 90.0% depending on the species, with a mean value of 59.6%. According to allele presence/absence of cross-species microsatellites, all the individuals representing 10 seahorse species could be accurately identified using one or more loci. In addition, we found both genome size and genetic distance between the source and target species negatively correlated with microsatellite cross-species amplification success. Moreover, a Neighbor-Joining dendrogram was constructed based on genetic similarity and suggested that the 10 seahorse species could be divided into four groups. These results demonstrate our method could improve the identification of seahorses, providing data to understand seahorse stock structure and recruitment dynamics, and therefore resources conservation.

Comparison of genetic and morphological characters in fossil teeth of grey voles from the Russian Far East (Rodentia: Cricetidae: Alexandromys)

Far-Eastern grey voles of the genus Alexandromys are a group of 12 species distributed exclusively in the Eastern Palearctic. Morphologically they are hard to be distinguished due to interspecific overlapping of several characters. In recent material of Alexandromys morphometric analyses of teeth (in particular the first lower molar) proved to be a good tool for species identification, although there are overlaps in several measurements. In subfossil and fossil material, preliminary analyses indicated considerable variation in tooth morphology and, in comparison to extant species, the presence of additional morphologically ambiguous forms. In the present study we tested in subfossil and fossil teeth of the genus Alexandromys from Russian Far East whether taxonomic classification based on morphology is in concordance with species determination based on DNA analysis. We also asked whether the morphologically ambiguous forms could be assigned to described species by means of DNA sequence analysis. The material originated from Medvezhyi Klyk Cave Deposits (Primorskyi Krai, Russia) and had an age of 2000–40,000 years. The results showed that the success rate in PCR amplification was ∼50% and it was possible to obtain PCR products of >400bp. The concordance between taxonomic assignment based on morphology and genetics was high. Out of 17 samples that were successful in the sequence analysis, only one yielded a classification that contradicts morphology and one specimen could not be classified since the delivered sequence most probably is a numt (nuclear copy of mitochondrial origin). Although only one of the four unidentified teeth in our sample was successful in the DNA analysis and allowed taxonomic assignment, our results suggest that genetic species determination could be a valuable tool to classify subfossil and fossil teeth in Arvicolinae.

Optimizing collection methods for noninvasive genetic sampling of neotropical felids

ABSTRACTField‐sampling methods for molecular scatology studies must be optimized, especially when working on elusive species in challenging tropical environments where rates of DNA degradation are elevated because of hot and humid weather conditions. To maximize polymerase chain reaction (PCR) amplification success and genotyping accuracy rates and to minimize genotyping error rates for fecal DNA samples of jaguars (Panthera onca) and co‐occurring Neotropical felids collected in Belize, Central America, we evaluated the performance of two fecal DNA storage techniques (dimethyl sulfoxide saline solution [DETs buffer] and 95% ethanol [EtOH]) suitable for long‐term preservation at remote tropical sites. Additionally, we tested fecal samples collected from 4 different locations on the scat (top, side, bottom, inside) at 2 different tropical forest types (tropical broadleaf and tropical pine forests). DETs buffer was the superior fecal DNA preservation method, with 44% higher PCR amplification success (P = 0.009) and 17% higher genotyping accuracy (P = 0.021) than 95% EtOH‐stored samples. Polymerase chain reaction amplification success of fecal DNA collected at the more open, pine‐forest (Pinus sp.) site differed significantly across locations on the scat, with highest mean success rates obtained from the top (85% ± 6.5% SD), followed by the side (79% ± 9.4% SD), bottom (76% ± 11.9% SD), and inside (69% ± 10.3% SD) of scat samples. Scat samples collected at the more closed‐canopy broadleaf site did not show any significant differences in amplification success rates across scat locations. We recommend that researchers optimize field‐sampling methods, including collection and storage protocols, by conducting a pilot study prior to their molecular scatology research efforts. © 2015 The Wildlife Society.

Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G.

Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.

Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba.

As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial assays.

CDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit.

BackgroundThe synthesis of complementary DNA (cDNA) for use in the detection of BCR-ABL1 at the Major Molecular Response (MMR) level is a well-established method used by clinical laboratories world-wide. However, the quality of cDNA provides sensitivity challenges and consequently affects the detection of Minimal Residual Disease (MRD).ResultsHerein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences.ConclusionsAccurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-015-0014-x) contains supplementary material, which is available to authorized users.