Low Sensitivity of Real Time PCRs Targeting Retrotransposon Sequences for the Detection of Schistosoma japonicum Complex DNA in Human Serum

While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.

Comparison of the Hybrid Capture II Method with a PCR-Based Screening Method Using a Carboxyfluorescein-Labeled Primer for Detecting Human Papillomavirus in Cervicovaginal Liquid-Based Cytology

Objective: Human papillomaviruses (HPVs) are DNA viruses, of which over 120 types have been identified. The main screening methods for HPV-DNA include the hybrid capture II (HC-II) and polymerase chain reaction (PCR) assays. Liquid-based cytology (LBC) is a high-quality technique developed to improve the diagnostic reliability of traditional Papanicolaou tests (Pap tests). However, relatively few studies have compared the efficacy of PCR and HC-II assays using cervicovaginal LBC specimens. In this study, we conducted a comparative analysis with results derived from the HC-II assay to assess whether a PCR-based assay using a novel carboxyfluorescein (FAM)-labeled primer could be applied to cervicovaginal LBC specimens. Methods and Results: We analyzed 59 specimens diagnosed as atypical squamous cells of undetermined significance (ASCUS) by Pap tests. After extracting DNA from cervicovaginal LBC specimens, we performed PCR using a FAM-labeled consensus primer, and then conducted fragment analysis to confirm the results. The value of the kappa statistic measuring the agreement between the PCR and HC-II results was 0.8557, or “almost perfect agreement.” Conclusion: Our novel HPV-PCR assay can be successfully applied to cervicovaginal LBC specimens for the detection of HPV subtypes.

Human Papillomavirus Genome based Detection and Typing: A Holistic Molecular Approach

Human Papillomavirus (HPV) is a species specific double-stranded DNA virus infecting human cutaneous or mucosal tissues. The genome structure of HPV is extremely polymorphic hence making it difficult to discriminate between them. HPV exhibits numerous dissimilar types that can be subdivided into high-risk (HR), probably high-risk and low-risk (LR), causing numerous types of cancers and warts around the genital organs in humans. Several screening methods are performed in order to detect cytological abnormalities and presence or absence of HPV genome. Currently available commercial kits and methods are designed to detect only a few HR/LR-HPV types, which are expensive adding to the economic burden of the affected individual and are not freely available. These gaps could be minimized through Polymerase Chain reaction (PCR) method, which is a gold standard and a cost-effective technique for the detection of most HPV (both known and unknown) types by using specific consensus primers in minimal lab setup. In this context, numerous studies have validated the effectiveness of different sets of consensus primers in the screening of HPVs. Numerous consensus primers, such as E6, E6/E7, GP-E6/E7, MY09/11, GP5+/GP6+, SPF10, and PGMY09/11 have been developed to detect the presence of HPV DNA. In addition, HPV detection sensitivity could be achieved through consensus primer sets targeting specific ORF regions like L1 and E6, which may finally assist in better diagnosis of several unknown HR-HPVs. The present review, provides a summary of the available methods, kits and consensus primer sets for HPV genome based detection, their advantages and limitations along with future goals to be set for HPV detection.

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

ABSTRACTHuman rhinoviruses (HRV) comprise 3 species representing more than 150 genotypes. As an important human respiratory pathogen, molecular detection is an indispensable tool for diagnosis and surveillance. However, the sequence diversity of HRV genotypes poses challenges for developing robust molecular methods that detect all genotypes with equal efficiencies. This study compares the accuracies of reverse transcription-quantitative PCR (RT-qPCR) and reverse transcription-digital PCR (RT-dPCR) for quantifying HRV RNA using genotype-specific primers and probes and a consensus primer/probe set targeting the 5′ noncoding region of HRV. When using consensus primers and probes for the quantification of HRV, RT-dPCR outperformed RT-qPCR by consistently and accurately quantifying HRV RNAs across more genotype groups, despite the presence of up to 2 target-sequence mismatches within the primer or probe binding region. Because it does not rely on amplification efficiency, which can be affected by sequence mismatches in primer/probe binding regions, RT-dPCR may be the optimal molecular method for future HRV quantification studies and for quantitating other viruses with high sequence diversity.

Phylogenetic relationships among the first and second introns of selected Prunus S-RNase genes

Rahemi, A., Gradziel T.M., Chaparro J.X., Folta, K.M., Taghavi, T., Fatahi, R., Ebadi, A. and Hassani, D. 2015. Phylogenetic relationships among the first and second introns of selected Prunus S-RNase genes. Can. J. Plant Sci. 95: 1145–1154. To identify and evaluate self-incompatible alleles in almonds and related germplasm, DNA from 15 Prunus species was amplified using two degenerate consensus primer pairs flanking first and second S-locus introns (PaConsI-FD+EM-Pc1ConsRD and EM-Pc2ConsFD+EM-Pc3ConsRD). Twenty-eight amplified PCR products were analyzed by automated sequencer capillary electrophoresis. Sequenced fragments were aligned against available Prunus S-locus sequences in the National Center for Biotechnology Information and S-alleles identities were determined. The phylogenetic relationships between S-alleles in the germplasm studied were determined by the homology between their sequences and dendrograms were obtained for each primer pair. The Maximum Likelihood (homology) ranged from 84 to 100%. Most sequences were similar to cultivated almond (Prunus dulcis) or to the European wild almond (P. webbii). Twenty-six alleles for the first and the second introns were registered in the database in the GenBank. Two sequences of the first and second introns, which were taken from Prunus nairica and had similarity in GenBank, were registered in the database under a common sequence of the first and second intron. Analysis of phylogenetic relationships (dendrograms) among S-alleles from wild almond species as well as S-alleles cluster relations showed most pairs of alleles well supported by bootstrap.

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.

Screening for Residual Disease in Pediatric Burkitt Lymphoma Using Consensus Primer Pools

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgVH) genes,IgVHprimer pools fromIgVH1–IgVH7regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of usingIgVHprimer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD usingIgVHprimer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.