Gene-specific Oligonucleotide Research Articles

Oligonucleotide Design and Construction of a Gene-Targeting CRISPR-Cas9 Plasmid in Escherichia coli for Generating a Gene-Deletion Strain in Staphylococcus aureus.

Gene deletions can be constructed in Staphylococcus aureus using recombineering in combination with a CRISPR-Cas9 counterselection approach. The method involves first designing the recombineering oligonucleotides and generating the relevant plasmids, and then introducing these elements into S. aureus to generate the desired gene deletion. Here, we describe the first part of this workflow, oligonucleotide design and plasmid generation. To better illustrate the method and oligonucleotide design, the construction of a 55-bp out-of-frame deletion in the S. aureus geh gene will be presented as a specific example. To this end, we describe the use of geh gene-specific recombineering oligonucleotides and the construction of a geh gene-targeting CRISPR-Cas9 plasmid. The protocol is divided into three parts: (1) design of the gene-specific targeting spacer oligonucleotides for introduction into the CRISPR-Cas9 plasmid pCas9-counter, (2) design of 90-mer recombineering oligonucleotides to generate a 55-bp out-of-frame gene deletion, and (3) construction of the gene-targeting CRISPR-Cas9 plasmid pCas9-geh, plasmid recovery in Escherichia coli, and confirmation by colony PCR and sequencing. The method can easily be adapted to design deletions for other S. aureus genes.

SETDB1 Modulates Degradation of Phosphorylated RB and Anticancer Efficacy of CDK4/6 Inhibitors.

The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates.

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis.

Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific CYP2E1 genetic variants. To design and evaluate the next-generation sequencing-based method for full-length CYP2E1 gene polymorphism analysis. Seven gene-specific oligonucleotide primer pairs targeting overlapping CYP2E1 gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform. The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the CYP2E1 gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy. Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the CYP2E1 gene are of clinical significance.

No Evidence of Influenza A Virus RNA in South African Backyard Swine in the uMgungundlovu District of the KwaZulu-Natal Province

Background: Influenza A virus (IAV) is the most widely reported influenza type in swine populations worldwide. Since no information was available on IAV prevalence in backyard swine populations in South Africa, we, for the first time, performed a molecular surveillance for detecting IAV prevalence in backyard swine in the uMgungundlovu District of the KwaZulu-Natal province of South Africa. Methods: Swine oral secretion (saliva) samples (n=102) were collected from three backyard farms distantly located in the uMgungundlovu District in March 2021. Total RNA was used to detect IAV using a one-step real-time RT-PCR assay with matrix gene-specific oligonucleotide primers and a TaqMan probe. Result: None of the samples amplified the IAV matrix gene suggesting that the swine under investigation were free from IAV active infection; however, the quantified viral RNA of swine saliva samples needs further investigation to determine the presence of other RNA viruses. The present study was conducted during COVID-19 related lockdown restrictions in South Africa, during which a significantly decreased influenza activity was reported in the Southern Hemisphere. Increased alertness and behavioural changes in people, such as maintaining hygiene and using a face mask for day-to-day operations, might have limited the transmission of IAV between humans and swine.

Multi-omics analysis to visualize the dynamic roles of defense genes in the response of tea plants to gray blight.

Gray blight (GB) is one of the most destructive diseases of tea plants, causing considerable damage and productivity losses; however, the dynamic roles of defense genes during pathogen infection remain largely unclear. To explore the numerous molecular interactions associated with GB stress in tea plants, we employed transcriptome, sRNAome and degradome sequencing from 1 to 13days post-inoculation (dpi) at 3-day intervals. The transcriptomics results showed that differentially expressed genes (DEGs) related to flavonoid synthesis, such as chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL), were particularly induced at 4 dpi. Consistent with this, the contents of catechins (especially gallocatechin), which are the dominant flavonoids in tea plants, also increased in the leaves of tea plants infected with GB. Combined analysis of the sRNAome and degradome revealed that microRNAs could mediate tea plant immunity by regulating DEG expression at the post-transcriptional level. Co-expression network analysis demonstrated that miR530b-ethylene responsive factor 96 (ERF96) and miRn211-thaumatin-like protein (TLP) play crucial roles in the response to GB. Accordingly, gene-specific antisense oligonucleotide assays suggested that suppressing ERF96 decreased the levels of reactive oxygen species (ROS), whereas suppressing TLP increased the levels of ROS. Furthermore, ERF96 was induced, but TLP was suppressed, in susceptible tea cultivars. Our results collectively demonstrate that ERF96 is a negative regulator and TLP is a positive regulator in the response of tea plants to GB. Taken together, our comprehensive integrated analysis reveals a dynamic regulatory network linked to GB stress in tea plants and provides candidate genes for improvement of tea plants.

Single cell epigenetic visualization assay.

Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.

Localisation of probiotic Vibrio midae K811 within the gastrointestinal tract of post-larval abalone, Haliotis midae

An artificial biofilm probiotic-supplemented diet was shown to enhance the growth and survival of post-larval South African abalone, Haliotis midae. To ensure the effectiveness of the probiotic bacterium Vibrio midae K811 in an aquaculture environment, we wished to investigate whether V. midae K811 reaches the abalone intestinal tract where it is expected to exert its probiotic effect. Thus, the aim of this study was to investigate the colonization potential of V. midae K811 and to localise the probiotic within the gastrointestinal tract of post-larval H. midae. The Tn9 cat gene incorporated in V. midae K811 was PCR amplified from total DNA isolated from whole abalone homogenates up to 10 days following the cessation of feeding abalone with the probiotic-supplemented diet, while viable V. midae K811 was detected in abalone homogenates up to 25 days after the post-larval abalone had been switched from an alginate surface supplemented with V. midae K811 to a surface supplemented with Cocconeis sp. Furthermore, in situ hybridization using two Tn9 cat gene-specific DIG-labelled oligonucleotide probes detected V. midae K811 in the intestine of post-larval H. midae, confirming that the probiotic reaches the digestive tract of post-larval abalone placed on an alginate biofilm supplemented with the probiotic, and that the bacterium can persist in the abalone digestive tract for a period of at least 3 weeks.

CRISPR-Cas12a-assisted PCR tagging of mammalian genes.

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.

Antibacterial Peptide Nucleic Acids-Facts and Perspectives.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.

Rapid Amplification of Sequences from the 3′ Ends of mRNAs: 3′-RACE

cDNAs whose first-strand synthesis has been primed by oligo(dT) generally contain the 3' sequences of the RNA. However, cDNAs generated by internal gene-specific primers will usually lack one or both terminal regions of the parental mRNA. And alternate splicing or the presence of an internal A-rich tract can generate cDNA clones that lack 3' sequences. These sequences can be recovered using 3'-RACE (rapid amplification of cDNA ends), which can also be used to map the 3' termini of families of mRNAs with alternative polyadenylation sites. Briefly, a population of mRNAs is transcribed into cDNA using an adaptor-primer with a poly(T) at its 3' end and 30-40 nt containing the recognition sites for two or three restriction enzymes at its 5' end. Reverse transcription is usually followed by two successive rounds of polymerase chain reaction (PCR). The first is primed by a gene-specific sense oligonucleotide and the antisense (dT) adaptor-primer. The products of the first PCR can be used as templates for a second, nested PCR, which is primed by a gene-specific sense oligonucleotide internal to the first, and a second antisense oligonucleotide whose sequence is identical to the central region of the (dT) adaptor-primer. The products amplified in the second PCR are isolated, cleaved with a suitable restriction enzyme, cloned, and characterized.

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Phylogenetic analyses of the genus Aeromonas based on housekeeping gene sequencing and its influence on systematics.

The phylogenies derived from housekeeping gene sequence alignments, although mere evolutionary hypotheses, have increased our knowledge about the Aeromonas genetic diversity, providing a robust species delineation framework invaluable for reliable, easy and fast species identification. Previous classifications of Aeromonas, have been fully surpassed by recently developed phylogenetic (natural) classification obtained from the analysis of so-called 'molecular chronometers'. Despite ribosomal RNAs cannot split all known Aeromonas species, the conserved nature of 16S rRNA offers reliable alignments containing mosaics of sequence signatures which may serve as targets of genus-specific oligonucleotides for subsequent identification/detection tests in samples without culturing. On the contrary, some housekeeping genes coding for proteins show a much better chronometric capacity to discriminate highly related strains. Although both, species and loci, do not all evolve at exactly the same rate, published Aeromonas phylogenies were congruent to each other, indicating that, phylogenetic markers are synchronized and a concatenated multigene phylogeny, may be 'the mirror' of the entire genomic relationships. Thanks to MLPA approaches, the discovery of new Aeromonas species and strains of rarely isolated species is today more frequent and, consequently, should be extensively promoted for isolate screening and species identification. Although, accumulated data still should be carefully catalogued to inherit a reliable database.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis

BackgroundEquine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay’s utility for regulatory testing.ResultsWe developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay’s performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5–90.7%), 90.5% (95% CI: 87.3–92.3%) and 87.4% (95% CI: 84.0–90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9–12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7–10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9–63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR.ConclusionsThe duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.

Enrichment of Viral Nucleic Acids by Solution Hybrid Selection with Genus Specific Oligonucleotides

Despite recent advances, our knowledge of potential and rare human pathogens is far from exhaustive. Current molecular diagnostic tools mainly rely on the specific amplification of marker sequences and may overlook infections caused by unknown and rare pathogens. Using high-throughput sequencing (HTS) can solve this problem; but, due to the extremely low fraction of pathogen genetic material in clinical samples, its application is only cost-effective in special, rather than routine, cases. In this study, we present a method for the semi-specific enrichment of viral conservative sequences in a HTS library by hybridization in solution with genus-specific degenerate biotinylated oligonucleotides. Nucleic acids of the test viruses (yellow fever virus and Japanese encephalitis virus) were enriched by solution hybrid selection using pan-flavivirus oligonucleotides. Moreover, enterovirus (family: Picornaviridae, genus: Enterovirus) sequences were successfully enriched using foot-and-mouth disease virus (family: Picornaviridae, genus: Aphthovirus) oligonucleotide. The enrichment factor relative to the background nucleic acid was about 1,000-fold. As hybridization has less stringent oligonucleotide match requirements than PCR, few oligonucleotides are sufficient to cover the potential sequence variation in the whole genus and may even enrich nucleic acids of viruses of other related genera. Efficient enrichment of viral sequences makes its use in diagnostics cost-efficient.

Cardiac troponin T is necessary for normal development in the embryonic chick heart.

The heart is the first functioning organ to develop during embryogenesis. The formation of the heart is a tightly regulated and complex process, and alterations to its development can result in congenital heart defects. Mutations in sarcomeric proteins, such as alpha myosin heavy chain and cardiac alpha actin, have now been associated with congenital heart defects in humans, often with atrial septal defects. However, cardiac troponin T (cTNT encoded by gene TNNT2) has not. Using gene‐specific antisense oligonucleotides, we have investigated the role of cTNT in chick cardiogenesis. TNNT2 is expressed throughout heart development and in the postnatal heart. TNNT2‐morpholino treatment resulted in abnormal atrial septal growth and a reduction in the number of trabeculae in the developing primitive ventricular chamber. External analysis revealed the development of diverticula from the ventricular myocardial wall which showed no evidence of fibrosis and still retained a myocardial phenotype. Sarcomeric assembly appeared normal in these treated hearts. In humans, congenital ventricular diverticulum is a rare condition, which has not yet been genetically associated. However, abnormal haemodynamics is known to cause structural defects in the heart. Further, structural defects, including atrial septal defects and congenital diverticula, have previously been associated with conduction anomalies. Therefore, to provide mechanistic insights into the effect that cTNT knockdown has on the developing heart, quantitative PCR was performed to determine the expression of the shear stress responsive gene NOS3 and the conduction gene TBX3. Both genes were differentially expressed compared to controls. Therefore, a reduction in cTNT in the developing heart results in abnormal atrial septal formation and aberrant ventricular morphogenesis. We hypothesize that alterations to the haemodynamics, indicated by differential NOS3 expression, causes these abnormalities in growth in cTNT knockdown hearts. In addition, the muscular diverticula reported here suggest a novel role for mutations of structural sarcomeric proteins in the pathogenesis of congenital cardiac diverticula. From these studies, we suggest TNNT2 is a gene worthy of screening for those with a congenital heart defect, particularly atrial septal defects and ventricular diverticula.

Molecular characterization and polymorphism detection in HSPB6 gene in Sahiwal cattle

The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphisms in the HSPB6 gene of Sahiwal cattle. A total of 100 Sahiwal cattle maintained at LRC, NDRI, Karnal (Haryana) were included in the study. HSPB6 gene has been mapped on Bos taurus autosome 18 (BTA 18) and spans nearly 2620 bp comprising 3 exons and 2 introns. Four sets of forward and reverse gene-specific oligonucleotide primers of HSPB6 gene were designed using Primer3 software and amplicon size of 529 bp, 460 bp, 670 bp and 502 bp were ascertained. On the basis of comparative sequence analysis, total three polymorphic loci (SNPs) were detected at locus G436A, A2152G and A2417T in HSPB6 gene as compare to Bos taurus (NCBI GenBank AC_000175.1). Out of these three SNPs, two (A2152G & A2417T) were found in 3′-UTR region and third one (G436A) was detected in intronic region 1. However, monomorphic pattern was found in exon 2 of HSPB6 gene in Sahiwal cattle. Further research need be directed to find out the association of allelic variants in major heat shock protein genes to establish markers for the future genetic improvement of livestock through marker assisted selection.

Production of virus-free garlic (Allium sativum L.) through meristem tip culture after solar or hot air treatment of cloves

SummaryGarlic (Allium sativum L.) is a clonally propagated crop that can be infected by many virus species which tend to accumulate year-after-year, leading to yield reductions and varietal degeneration. Therefore, the production and use of virus-free planting material is vital to sustain productivity. In this study, Onion yellow dwarf virus (OYDV; Potyvirus), Garlic common latent virus (GarCLV; Carlavirus), Shallot latent virus (SLV; Carlavirus), and Garlic virus X (GarV-X; Allexivirus) were detected in cloves of the commercial garlic cultivar, ‘G-1’ (‘Jamuna Safed’) by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). DAC-ELISA was performed using specific antisera against each virus species, and confirmed through reverse transcription-polymerase chain reactions (RT-PCR) using virus or genus-specific oligonucleotide primers. To obtain virus-free plants of ‘G-1’, cloves were subjected to solar heat treatment for 5, 10, or 15 d, or to hot air treatment (at 37°C, 40°C, or 42°C for 7, 14, or 21 d) combined with meristem isolation and in vitro culture, then indexed for all viruses using DAC-ELISA and RT-PCR. Meristems from solar heat-treated or hot air-treated cloves regenerated after 90 d and 45 d in culture, respectively. Solar heat treatment for 15 d resulted in 17 - 18% regeneration, and all were free from GarCLV, SLV, and OYDV. However, when solar heat-treated for 10 d, only 66% of the 17 - 18% regenerated plants were free from all viruses, including GarV-X. Hot air treatment at 37°C or 40°C eliminated GarCLV, SLV, and OYDV in 20 - 38% and 42 - 62% of regenerated plants, respectively, for the different durations of exposure. However, GarV-X could not be eliminated from any of the regenerated plants by hot air treatment, irrespective of the duration of exposure. Meristems from cloves that had been hot air-treated at 42°C resulted in 50 - 66% regeneration, of which 53 - 57% were free from GarCLV, SLV, and OYDV when treated for 7 d or 14 d, and 29% were free from all viruses when treated for 21 d. For most treatments, an increase in the duration of exposure to heat resulted in reduced regeneration, but increased the rate of recovery of virus-free plants. To our knowledge, this is the first study to report the use of solar treatment for virus elimination in garlic and the development of an efficient tissue culture protocol for the production of virus-free garlic plants using different thermotherapy treatments coupled with meristem tip culture.

Keeping qRT-PCR rigorous and biologically relevant.

Because of the importance of differential gene expression in the growth, development, and reproduction of organisms, assays of mRNA transcript levels are widely employed in biological research. The most frequently used assay combines reverse transcription (RT) with quantitative polymerase chain reaction (qPCR) and is known as qRT-PCR (or RT-qPCR). In practice, RT generates a single-stranded cDNA copy of each molecule in an mRNA population, after which a qPCR thermocycler is used in conjunction with gene-specific oligonucleotide primers to amplify selected target cDNAs (Bustin et al. 2009). Fluorescent probes allow progress to be monitored through three phases: an initial phase during which the increasing signal cannot be distinguished from background noise, a 2nd phase of observable exponential signal increase, and a third and final phase of slowing signal increase as reagents and other factors become rate limiting. Quantification of each target mRNA requires the determination of the number of cycles (known as Ct, Cq, Cp, or TOP) required to reach a pre-set fluorescence threshold during the second phase. When a transcript is twice as abundant in one sample as it is in another, the Ct will occur about one cycle earlier for the more abundant transcript. The varying transcript levels of target genes must then be normalized against the stable transcript levels of one or more reference genes present in the same mRNA population. Algorithms are available to validate the stability of these reference genes. With use of appropriate controls, standardization, and normalization, measurements of Ct can lead to reliable estimates of the relative amount of a target transcript in the input mRNA population (Bustin et al. 2009). Despite its complexity, qRT-PCR is rapid and cost effective, and, if sufficient care is taken with the design and execution of experiments, it is reliable and sensitive. There are, however, two trends of concern in the publication of qRT-PCR experiments. The first is that many papers do not contain enough information to judge whether the data are reliable. Bustin et al. (2009) addressed this question by defining ‘‘minimum information for publication of quantitative real-time PCR experiments’’ (MIQE). The second trend of concern is the increasing number of manuscripts that claim to have identified reference genes without comparing them to target genes in a well-defined and relevant biological context. We agree with Bustin et al. (2010), who considered it pointless and misleading to publish such ‘reference gene papers’ as stand-alone reports. In response to these trends, we propose that publishable qRT-PCR manuscripts should satisfy two criteria. First, the design, execution, and reporting of qRT-PCR experiments should address the items listed in the MIQE guidelines. Second, the selection and validation of reference genes should be reported in relation to target gene behavior in a well-defined biological context. Here we discuss the background to these criteria and their implementation relative to plant biology studies. Communicated by Neal Stewart.

Full Genotyping of a Highly Polymorphic Human Gene Trait by Time-Resolved Fluorescence Resonance Energy Transfer

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52–57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52–57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52–57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.