Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters.

Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of Taq DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines Escherichia coli serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies KRAS mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.

Protein Dynamic Landscape of Pig Embryos during Peri-Implantation Development.

Properly developed embryos are critical for successful embryo implantation. The dynamic landscape of proteins as executors of biological processes in pig peri-implantation embryos has not been reported so far. In this study, we collected pig embryos from days 9, 12, and 15 of pregnancy during the peri-implantation stage for a PASEF-based quantitative proteomic analysis. In total, approximately 8000 proteins were identified. These proteins were classified as stage-exclusive proteins and stage-specific proteins, respectively, based on their presence and dynamic abundance changes at each stage. Functional analysis showed that their roles are consistent with the physiological processes of corresponding stages, such as the biosynthesis of amino acids and peptides at P09, the regulation of actin cytoskeletal organization and complement activation at P12, and the vesicular transport at P15. Correlation analysis between mRNAs and proteins showed a general positive correlation between pig peri-implantation embryonic mRNAs and proteins. Cross-species comparisons with human early embryos identified some conserved proteins that may be important in regulating embryonic development, such as STAT3, AP2A1, and PFAS. Our study provides a comprehensive overview of the pig embryo proteome during implantation, fills gaps in relevant developmental studies, and identifies some important proteins that may serve as potential targets for future research.

Accurate Detection of Multiple Tumor Mutations in Formalin-Fixed Paraffin-Embedded Tissues by Coupling Sequence Artifacts Elimination and Mutation Enrichment With MeltArray.

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary source of DNA for companion diagnostics (CDx) of cancers. Degradation of FFPE tissue DNA and inherent tumor heterogeneity constitute serious challenges in current CDx assays. To address these limitations, we introduced sequence artifact elimination and mutation enrichment to MeltArray, a highly multiplexed PCR approach, to establish an integrated protocol that provides accuracy, ease of use, and rapidness. Using PIK3CA mutations as a model, we established a MeltArray protocol that could eliminate sequence artifacts completely and enrich mutations from 23.5- to 59.4-fold via a single-reaction pretreatment step comprising uracil-DNA-glycosylase excision and PCR clamping. The entire protocol could identify 13 PIK3CA hotspot mutations of 0.05% to 0.5% mutant allele fractions within 5 hours. Evaluation of 106 breast cancer and 40 matched normal FFPE tissue samples showed that all 47 PIK3CA mutant samples were from the cancer tissue, and no false-positive results were detected in the normal samples. Further evaluation of 105 colorectal and 40 matched normal FFPE tissue samples revealed that 11 PIK3CA mutants were solely from the cancer sample. The detection results of our protocol were consistent with those of the droplet digital PCR assays that underwent sequence artifact elimination. Of the 60 colorectal samples with next-generation sequencing results, the MeltArray protocol detected 2 additional mutant samples with low mutant allele fractions. We conclude that the new protocol provides an improved alternative to current CDx assays for detecting tumor mutations in FFPE tissue DNA.

Multiplex Asymmetric PCR by Combining the Amplification Refractory Mutation System with the Homo-Tag-Assisted Nondimer System.

Asymmetric PCR is widely used to produce single-stranded amplicons (ss-amplicons) for various downstream applications. However, conventional asymmetric PCR schemes are susceptible to events that affect primer availability, which can be exacerbated by multiplex amplification. In this study, a new multiplex asymmetric PCR approach that combines the amplification refractory mutation system (ARMS) with the homo-Tag-assisted nondimer system (HANDS) is described. ARMS-HANDS (A-H) PCR utilizes equimolar-tailed forward and reverse primers and an excess Tag primer. The tailed primer pairs initiate exponential symmetric amplification, whereas the Tag primer drives linear asymmetric amplification along fully matched strands but not one-nucleotide mismatched strands, thereby generating excess ss-amplicons. The production of ss-amplicons is validated using agarose gel electrophoresis, sequencing, and melting curve analysis. Primer dimer alleviation is confirmed by both the reduced Loss function value and a 20-fold higher sensitivity in an 11-plex A-H PCR assay than in an 11-plex conventional asymmetric PCR assay. Moreover, A-H PCR demonstrates unbiased amplification by its allele quantitative ability in correct identification of all 31 trisomy 21 samples among 342 clinical samples. A-H PCR is a new generation of multiplex asymmetric amplification approach with various applications, especially when sensitive and quantitative detection is required.

Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05-0.5% within 2 h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.

Uncovering the function of insulin receptor substrate in termites' immunity through active immunization.

Insulin receptor substrate (IRS) proteins are key mediators in insulin signaling pathway. In social insect lives, IRS proteins played important roles in caste differentiation and foraging, but there function in disease defenses such as active immunization has not been reported yet. To investigate the issue, we successfully suppressed the IRS gene 3 days after dsRNA injection. Suppressing IRS gene increased the contents of glucose, trehalose, glycogen, and triglyceride and decreased the content of pyruvate in termites, and led to the metabolic disorder of glucose and lipids. IRS suppressing significantly enhanced grooming behaviors of nestmates of fungus-contaminated termites and hence increased the conidial load in the guts of the nestmates. Additionally, IRS suppressing led to significant downregulation of the immune genes Gram-negative bacteria-binding protein2 (GNBP2) and termicin and upregulation of the apoptotic gene caspase8, and hence diminished antifungal activity of nestmates of fungus-contaminated termites. The above abnormal behavioral and physiological responses significantly decreased the survival rate of dsIRS-injected nestmates of the fungus-contaminated termites. These findings suggest that IRS is involved in regulation of active immunization in termites, providing a better understanding of the link between insulin signaling and the social immunity of termites.

Entomopathogenic fungal infection following immune gene silencing decreased behavioral and physiological fitness in Aedes aegypti mosquitoes.

Entomopathogenic fungi are a promising category of biocontrol agents with mosquitocidal properties. Prior studies have proved their potential to reduce fecundity, human biting and vector competence, all of them together determine vectorial capacity of the mosquitoes. Unfortunately, conventional vector control strategies are inadequate with growing problem of insecticide resistance and environmental deterioration. Therefore, alternate vector control measures are immediately needed and to accomplish that, an improved understanding of behavioral and physiological defense mechanisms of the mosquitoes against fungal infection is essential. In this study, fitness was considered with respect to different behavioral (self-grooming and flight), physiological (antifungal activity and antimicrobial peptides) parameters and survival rates as compared to the control group. We found a significant upregulation in CLSP2, TEP22, Rel1 and Rel2 genes at multiple time periods of fungal infection, which indicates the successful fungal infection and activation of Toll and IMD pathways in mosquitoes. RNAi-mediated silencing of Rel1 and Rel2 genes (transcription factors of Toll and IMD pathways, respectively) significantly reduced the survival, self-grooming frequencies and durations, and flight locomotor activity among adult Ae. aegypti female mosquitoes. Moreover, Rel1 and Rel2 knockdown significantly decreased antifungal activity and antimicrobial peptides expression levels in target mosquitoes. These results indicate an overall decrease in fitness of the mosquitoes after fungal challenge following Rel1 and Rel2 silencing. These findings provide an improved understanding of behavioral and physiological responses in mosquitoes with altered immunity against entomopathogenic fungal infections which can guide us towards the development of novel biocontrol strategies against mosquitoes.

The cGMP-dependent protein kinase gene can regulate trail-following behaviour and locomotion in the termite Reticulitermes chinensis Snyder.

Social behaviours in termites are closely related to the chemical communication between individuals. It is well known that foraging worker termites can use trail pheromones to orient and locomote along trails so as to take food resources back to the nest. However, it is still unclear how termites recognize trail pheromones. Here, we cloned and sequenced the cGMP-dependent protein kinase (PKG) gene from the termite Reticulitermes chinensis Snyder, and then examined the response of termites to trail pheromones after silencing PKG through RNA interference. We found that PKG knockdown impaired termite ability to follow trail pheromones accurately and exhibited irregular behavioural trajectories in response to the trail pheromone in the termite R. chinensis. Our locomotion assays further showed that PKG knockdown significantly increased the turn angle and angular velocity in the termite R. chinensis. These findings help us better understanding the molecular regulatory mechanism of foraging communications in termites.

A fungal sirtuin modulates development and virulence in the insect pathogen, Beauveria bassiana.

Chromatin transitions are mediated in part by acetylation/deacetylation post-translational modifications of histones. Histone deacetylases, e.g. sirtuins (Sir-proteins), repress transcription via promotion of heterochromatin formation. Here, we characterize the Sir2 class III histone deacetylase (BbSir2) in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. BbSir2 is shown to contribute to the deacetylation of lysine residues on H3 and H4 histones. Targeted gene knockout of BbSir2 resulted in impaired asexual development, reduced abilities to utilize various carbon/nitrogen sources, reduced tolerance to oxidative, heat, and UV stress, and attenuated virulence. ΔBbSir2 cells showed disrupted cell cycle development and abnormal hyphal septation patterns. Proteomic protein acetylation analyses of wild type and ΔBbSir2 cells revealed the differential abundance of 462 proteins and altered (hyper- or hypo-) acetylation of 436 lysine residues on 350 proteins. Bioinformatic analyses revealed enrichment in pathways involved in carbon/nitrogen metabolism, cell cycle control and cell rescue, defence and mitochondrial functioning. Critical targets involved in virulence included LysM effector proteins and a benzoquinone oxidoreductase implicated in detoxification of cuticular compounds. These data indicate broad effects of BbSir2 on fungal development and stress response, with identification of discrete targets that can account for the observed (decreased) virulence phenotype.

Potential role of lysine succinylation in the response of moths to artificial light at night stress.

Artificial light at night (ALAN) is a widespread environmental pollutant and stressor. Many nocturnal insects have been shown to experience ALAN stress. However, few studies have been conducted to uncover the mechanism by which nocturnal insects respond to ALAN stress. Previous studies suggest that lysine succinylation (Ksuc) is a potential mechanism that coordinates energy metabolism and antioxidant activity under stressful conditions. Mythimna separata (Walker) (M. separata) is a nocturnal insect that has been stressed by ALAN. In this study, we quantified the relative proteomic Ksuc levels in ALAN-stressed M. separata. Of the 466 identified Ksuc-modified proteins, 103 were hypersuccinylated/desuccinylated in ALAN-stressed moths. The hypersuccinylated/desuccinylated proteins were shown to be involved in various biological processes. In particular, they were enriched in metabolic processes, reactive oxygen species (ROS) homeostasis and the neuromuscular system. Furthermore, we demonstrated that Ksuc might affect moth locomotion by intervening with and coordinating these systems under ALAN stress. These findings suggest that Ksuc plays a vital role in the moth response to ALAN stress and moth locomotion behavior and provide a new perspective on the impact of ALAN on nocturnal insect populations and species communities.

The bromodomain protein Brd4 insulates chromatin from DNA damage signalling.

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.

The Influence of Allogrooming Behavior on Individual Innate Immunity in the Subterranean Termite Reticulitermes chinensis (Isoptera: Rhinotermitidae).

Insect societies have evolved a series of disease defenses against pathogens, including social sanitary behavior and individual innate immunity. However, whether sanitary behavior can affect individual innate immunity remains unknown. Here, we exposed the termite Reticulitermes chinensis Snyder to the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin(Ascomycota: Hypocreales), and then measured their allogrooming behavior, conidia load, infection mortality, antifungal activity and immune gene expressions . Our results showed that most of the fungal conidia were fast removed from the cuticles of the grouped termites by intensive allogrooming behavior, resulting in low mortality. The antifungal activity and immune gene expressions (termicin and transferrin) in grouped exposed termites were significantly lower than those in single exposed termite but not significantly different from those in unexposed treatments. These results suggest that allogrooming behavior can fast remove fungal conidia from termite cuticles and then decrease their physiological investment in individual innate immunity.

Correction to: Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

Following publication of the original article [1], the authors reported an error in Table 3 on page 4. Variant No. 18 should be " p.Ser339Phe c.1016C>T " (as given in Number 117 of Additional file 2).

Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

BACKGROUND: Phenylketonuria (PKU), which primarily results from a deficiency of phenylalanine hydroxylase (PAH), is one of the most common inherited inborn errors of metabolism that impairs postnatal cognitive development. The incidence of various PAH variations differs by race and ethnicity. The aim of the present study was to characterize the PAH gene variants of a Han population from Northern China. METHODS: In total, 655 PKU patients and their families were recruited for this study; each proband was diagnosed both clinically and biochemically with phenylketonuria. Subjects were sequentially screened for single-base variants and exon deletions or duplications within PAH via direct Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS: A spectrum of 174 distinct PAH variants was identified: 152 previously documented variants and 22 novel variants. While single-base variants were distributed throughout the 13 exons, they were particularly concentrated in exons 7 (33.3%), 11 (14.2%), 6 (13.2%), 12 (11.0%), 3 (10.4%), and 5 (4.4%). The predominant variant was p.Arg243Gln (17.7%), followed by Ex6-96A > G (8.3%), p.Val399 = (6.4%), p.Arg53His (4.7%), p.Tyr356* (4.7%), p.Arg241Cys (4.6%), p.Arg413Pro (4.6%), p.Arg111* (4.4%), and c.442-1G > A (3.4%). Notably, two patients were also identified as carrying de novo variants. CONCLUSION: The composition of PAH gene variants in this Han population from Northern China was distinct from those of other ethnic groups. As such, the construction of a PAH gene variant database for Northern China is necessary to lay a foundation for genetic-based diagnoses, prenatal diagnoses, and population screening.

Rapid detection of G6PD mutations by multicolor melting curve analysis.

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured Tm was <0.3°C. The limit of detection was 1.0ng/µL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings.

Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis.

BACKGROUND: α-Thalassemia, caused by mutations in the α-globin genes, is one of the most common monogenic inherited disorders in the world. However, non-deletional α-thalassemia mutations remain undetected in routine clinical testing due to the lack of a suitable method. In this study, a closed- and single-tube assay for the detection of six common non-deletional α-thalassemia mutations in the HBA2 gene was developed based on multicolor melting curve analysis. METHODS: The assay consisted of one pair of primers specific for the HBA2 gene and four dual-labeled, self-quenched probes targeting six non-deletional α-thalassemia mutations. The sensitivity, reproducibility, and accuracy of the method were validated via 700 genomic DNA samples. RESULTS: The assay had a reproducibility of 100%, could detect gDNA of different genotype as low as 1 ng per reaction, and had an overall accuracy of 100% when compared with RDB analysis and Sanger sequencing. CONCLUSIONS: The developed assay is rapid, robust, and cost-effective while maintaining high sensitivity, specificity, and throughput.

Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection.

Multiplex analytical systems that allow detection of multiple nucleic acid targets in one assay can provide rapid characterization of a sample while still saving cost and resources. However, few systems have proven to offer a solution for mid-plex (e.g. 10- to 50-plex) analysis that is high throughput and cost effective. Here we describe the combined use of fluorescence color and melting temperature (Tm) as a virtual 2D label that enables homogenous detection of one order of magnitude more targets than current strategies on real-time polymerase chain reaction platform. The target was first hybridized with a pair of ligation oligonucleotides, one of which harbored an artificial sequence that had a unique Tm when hybridized with a reporter fluorogenic probe. The ligated products were then amplified by a universal primer pair and denatured by a melting curve analysis procedure. The targets were identified by their respective Tm values in the corresponding fluorescence detection channels. The proof-of-principle of this approach was validated by genotyping 15 high-risk human papillomaviruses and 48 human single-nucleotide polymorphisms. The robustness of this method was demonstrated by analyzing a large number of clinical samples in both cases. The combined merits of multiplexity, flexibility and simplicity should make this approach suitable for a variety of applications.

[Clinical evaluation of a melting curve analysis-based PCR assay for glucose phosphate dehydrogenase gene mutation detection].

OBJECTIVE: To evaluate the clinical value of multicolor melting curve analysis(MMCA) for detecting genetic mutations in G6PD deficiency. METHODS: A total of 402 peripheral blood samples(256 males and 146 females) were collected from suspected patients or their relatives at the Prenatal Diagnosis Center of Liuzhou Maternal and Child Health Hospital between March 2012 and May 2012. The samples were screened by G6PD/6PGD quantitative ratio testing. The reliability of the assay was evaluated by multiplex probe melting curve assay(which can detect 16 G6PD mutations) and DNA sequencing through a double blind study. RESULTS: One hundred seventy cases with G6PD/6PGD ratio < 1.0 and 232 cases with G6PD/6PGD ratio ≥ 1.0 were detected by the enzymological method. DNA sequencing has identified 182 wild type samples, 151 hemizygous mutation samples, 5 female homozygous mutation samples, 54 female heterozygous mutation samples and 10 female double heterozygous mutation samples. Multicolor melting curve analysis has detected 185 wild type samples, 148 hemizygous mutation samples, 5 female homozygous mutation samples, 55 female heterozygous mutation samples and 9 female double heterozygous mutation samples. The specificity and sensitivity of G6PD gene mutation detection by multicolor melting curve analysis were 100%(182/182) and 98.6%(217/220), respectively. The positive predictive value and negative predictive value were 99.5%(216/217) and 98.4%(182/185), respectively, and the Youden's index was 0.986. The concordance rate of the sample detection between the melting curve assay and DNA sequencing was 99.0%(398/402). Twenty-one different genotypes were detected by the multicolor melting curve analysis and 24 different genotypes were detected by DNA sequencing. Four samples containing mutations(c.196T>A or c.406C>T) were not detected by multicolor melting curve analysis, which can be attributed to different technical settings of the two methods. CONCLUSION: Multicolor melting curve analysis for G6PD gene mutation detection is a simple, rapid, sensitive and specific method, which can be used for clinical diagnosis of G6PD deficiency.

Accessory gland protein regulates pairing process and oviposition in the subterranean termite Reticulitermes chinensis after swarming.

Swarming and pairing behaviors are significant to population dispersal of termites. Tandem running is a key process in pairing behavior of dealates to find a mate. Succinylation can lead to significant changes in protein structure and function, which is widely involved in metabolism and behavior regulation in many organisms. However, whether succinylation modification regulates termites' tandem running is currently unknown. In this research, we performed quantitative modified proteomics of the subterranean termite Reticulitermes chinensis Snyder before and after alate swarming. The succinylation levels of accessory gland protein (ACP) were significantly altered after alate swarming. We found that ACP is enriched in male accessory gland and female oocytes of termites. The acetylation and succinylation sites of ACP affected tandem running of dealates. The transcriptome and metabolome analyses of alates injected with ACP and its mutant proteins showed that ß-alanine metabolism pathway was the major downstream pathway of ACP. Silencing the significantly differentially expressed genes in the ß-alanine metabolic pathway (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyisobutyrate dehydrogenase, methylmalonate-semialdehyde dehydrogenase) suppressed tandem running and altered oviposition of paired dealates. These findings demonstrate that protein translation modification is an important regulator of tandem running behavior of termites, which implies that the succinylation and acetylation modification sites of ACP could be potential targets for insecticide action. Our research offers a potential approach for developing novel dispersal inhibitors against social insect pests.