Melting Curve Research Articles

High resolution melting of chloroplast mini-barcode in star anise (Illicium verum) authentication

Star anise (Illicium verum), dubbed the ‘King of Spices’, is one of the most popular spices widely used in cuisine, cosmetics and herbal medicine. However, recent surveys have found that commercial products occasionally replace star anise with some toxic lookalike species. To address this problem, we applied a high resolution melting of chloroplast mini-barcode method to distinguish star anise from poisonous adulterants. We sequenced the complete chloroplast genomes of four reported adulterant species (i.e., Illicium lanceolatum, Illicium simonsii, Illicium henryi and Illicium brevistylum). We carried out a comparative analysis between these species and the published chloroplast genome of star anise. Although the chloroplast genomes are conserved among Illicium species, we found some divergent hotspot regions that can potentially exploit high resolution DNA mini-barcodes for species authentication. Subsequently, we used high resolution melting (HRM) assay to generate standard melting profiles of these mini-barcodes for these species. The melting data showed that the mini-barcodes derived from two divergence regions (i.e., atpF–atpH and ndhF–rpl32) have high resolution for species discrimination and could clearly distinguish star anise from four tested adulterants. We applied these two mini-barcodes to check twelve commercial products alleged or labelled as “star anise”, which were purchased from China traditional markets and internet shops. The HRM curves of eleven samples are clustered with the reference star anise with confidence higher than 95%, indicating that these products contain the correct species as declared. We also found one toxic adulterant, as its melting curve clustered with Illicium simonsii. We conclude that DNA mini-barcode derived from divergent hotspot region of chloroplast genome combined with HRM analysis is an effective and rapid strategy to discriminate star anise from its toxic lookalike adulterants.

Effect of WSSV-Infection on Relative Expression of Hemocyanin Genes in Litopenaeus Vannamei (Boone, 1931)

A Litopenaeus vannamei injected with the serum from the shrimps infected with WSSV-showed the typical symptoms of white spot syndrome at 48hpi. These include lethargy, lack of appetite, reduced and disoriented swimming activity. The negative control and serum from healthy shrimps did not show any band and the test samples contained a band corresponding to 310 bp which was like that of the positive control. Thus, the test samples were confirmed positive for the WSSV-infection. The mean serum protein concentration in healthy shrimp was 86.4 ± 6.3 mg/ml. on the other hand the serum protein concentration of WSSV-injected shrimps declined linearly and significantly to 58.8 ± 3.9, 46.2 ± 5.32, and 38.7 ± 6.7 mg/ml at 12, 24, and 48 hpi, respectively. Relative quantity of hemocyanin gene (I) expression in WSSV-injected shrimps increased gradually from 6 to 48 hpi. Analyses of relative quantification of hemocyanin gene (II) in the experimental groups showed that the relative quantity in WSSV- injected shrimps increased from 6 hpi up to 24 hpi, whereas at 48 hpi the WSSV-injected shrimps showed a decrease in relative quantity of hemocyanin gene (II). Analyses of relative quantification of hemocyanin gene (III) in the experimental groups showed that the relative quantity in WSSV-injected shrimps increased slowly from 6 to 12 hpi and the increase was found to be high at 24 and 48 hpi. The relative quantity increased in sham-injected shrimps. The melt curve analysis showed two peaks corresponding to 18S rRNA and hemocyanin gene I, II and III, respectively. The objective of the present study was directed towards the relative quantification of the hemocyanin genes (I, II and III) in L. vannamei upon WSSV-infection.

High Pressure Melting Curve of Fe Determined by Inter‐Metallic Fast Diffusion Technique

AbstractThe heat extracted from the core by the overlying mantle across the core‐mantle boundary controls the thermal evolution of the core. This in turn leads to the solidification of the inner core in association with the exsolution of light alloying elements into the liquid outer core. Although the temperature (T) at the inner core boundary (ICB) would be adjusted to account for the effects of the light elements, the melting T of Fe places an upper bound at the ICB and it is a vital point in the thermal profile of the core. Here, we determine the melting T of Fe in the multi‐anvil press by characterizing the interface of Fe‐W interaction. Our data place a tighter constraint on the melting curve of Fe between 8 and 21 GPa, that is directly applicable to small planetary bodies and serves as an anchor for melting curve of Fe at higher pressure.

DSC Melting Profile of Cold-Pressed Hemp Seed Oil as an Authenticity Fingerprint Influenced by Scanning Rate

Among the variety of edible cold-pressed oils on the market, hemp seed oil is becoming increasingly popular among scientists and consumers due to its plethora of nutritional compounds. In this study, the goal was to examine the thermal characteristics of cold-pressed hemp seed oil pressed from seeds of the Henola cultivar procured by five different suppliers in two different seasons. This aim of this research was to establish how various scanning rates can affect the unique thermal profile of cold-pressed hemp seed oil in terms of an authenticity assessment. The melting transition was manifested by curves with four peaks for all hemp seed oils; however, they differed for each scanning rate in terms of the shape and peak intensity. Comparing the curves obtained at heating rates of 1 and 2 °C/min, noticeable differences were observed in the melting transition parameters between hemp seed oils, showing that small differences in fatty acid composition can cause changes in DSC profiles. In contrast, at a scanning rate 5 °C/min, the melting curves were similar for all hemp seed oils. It was also observed that for all the scanning rates, there was a strong negative correlation between the total content of polyunsaturated fatty acids (ƩPUFAs) and the peak temperature of the three peaks (Tm2, Tm3, and Tm4). The most abundant fatty acids were PUFAs, i.e., linoleic acid (C18:2), with contents ranging from 47 to 55%; and α-linolenic acid (C 18:3 n–3), with contents ranging from 17 to 25%. The application of linear discriminant analysis (LDA) enabled a discriminant model to be built based on the DSC data obtained for differentiation of oils pressed from fresh and stored seeds.

A novel study on bean common mosaic virus accumulation shows disease resistance at the initial stage of infection in Phaseolus vulgaris.

Accurate and early diagnosis of bean common mosaic virus (BCMV) in Phaseolus vulgaris tissues is critical since the pathogen can spread easily and have long-term detrimental effects on bean production. The use of resistant varieties is a key factor in the management activities of BCMV. The study reported here describes the development and application of a novel SYBR Green-based quantitative real-time PCR (qRT-PCR) assay targeting the coat protein gene to determine the host sensitivity to the specific NL-4 strain of BCMV. The technique showed high specificity, validated by melting curve analysis, without cross-reaction. Further, the symptoms development of twenty advanced common bean genotypes after mechanical BCMV-NL-4 infection was evaluated and compared. The results showed that common bean genotypes exhibit varying levels of host susceptibility to this BCMV strain. The YLV-14 and BRS-22 genotypes were determined as the most resistant and susceptible genotypes, respectively, in terms of aggressiveness of symptoms. The accumulation of BCMV was analyzed in the resistant and susceptible genotypes 3, 6, and 9days following the inoculation by the newly developed qRT-PCR. The mean cycle threshold (Ct) values showed that the viral titer was significantly lower in YLV-14, which was evident in both root and leaf 3days after the inoculation. The qRT-PCR thus facilitated an accurate, specific, and feasible assessment of BCMV accumulation in bean tissues even in low virus titers, allowing novel clues in selecting resistant genotypes in the early stages of infection, which is critical for disease management. To the best of our knowledge, this is the first study of a successfully performed qRT-PCR to estimate BCMV quantification.

P–T Phase Diagram of Phosphorus Revisited

Melting and phase transitions of phosphorus allotropes have been studied in the 2–11 GPa pressure range using in situ electrical resistivity measurements and synchrotron X-ray diffraction. It was found that the melting curve of rhombohedral phosphorus exhibits a positive slope of 58(2) K/GPa and crosses the rhombohedral–simple cubic phase boundary (negative slope of −420(70) K/GPa) at about 8.3 GPa and 1490 K. The melting slope of simple cubic phosphorus can be estimated to be +300(90) K/GPa. Calculations of phosphorus phase equilibria have been performed using our experimental and literature data within the framework of the phenomenological thermodynamics. The constructed equilibrium p–T phase diagram accurately describes the experimentally observed phase relations in solid and liquid phosphorus up to 12 GPa and 2000 K.

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high-resolution melting assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected during the Alpha, Delta and Omicron [BA.1] waves of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities.

Accurate Identification of Leishmania Parasites in Sand Flies by Polymorphism Analysis of Cytochrome Oxidase Subunit 2 Gene Using Polymerase Chain Reaction and Quantitative PCR-High Resolution Melting Techniques in Iranian Border with Iraq.

Firmly identification of Leishmania in Phlebotomus papatasi and understanding of natural transmission cycles of parasites in sand flies are important for treatment and local control. Modified and developed method of High Resolution Melting (HRM) as a preferable technique was employed to accurate identification of Leishmania in sand flies from Iranian border with Iraq, by targeting cytochrome oxidase II (COII) gene and designing suitable primers. PCR products cloned into pTG19-T vector, then purified plasmid concentration was measured at 260 and 280nm wavelength. The melting curve plots were generated and DNA sequences were analyzed using Sequencher 3.1.1, CLC Main Workbench 5.5, MEGA 6, DnaSP5.10.01 and MedCalc® version 13.3.3 soft wares. Among about 3000 collected sand flies, 89 female Ph. papatasi were identified and two with L. major. In amplified fragment of COII gene among 611bp, 452bp had no genetic variations with low polymorphic sites (P= 0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). Leishmania major was discriminated in Ph. papatasi with 0.84 °C melting temperature (Tm) and unique curve based on thermodynamic differences was an important criterion using HRM technique. Subsequent war in Iraq made a high risk habitat for parasites transmission. It is important to discover accurate diagnostic procedures for leishmaniasis control.

MeltR software provides facile determination of nucleic acid thermodynamics

Thermodenaturation (melting) curves of macromolecules are used to determine folding thermodynamic parameters. Notably, this insight into RNA and DNA stability underlies nearest neighbor theory and diverse structure prediction tools. Analysis of UV-detected absorbance melting curves is complex and multivariate, requiring many data preprocessing, regression, and error analysis steps. The absorbance melting curve-fitting software MeltWin, introduced in 1996, provided a consistent and facile melting curve analysis platform used in a generation of folding parameters. Unfortunately, MeltWin software is not maintained and relies on idiosyncratic choices of baselines by the user. Herein, we provide MeltR, an open-source, curve-fitting package for analysis of macromolecular thermodynamic data. The MeltR package provides the facile conversion of melting curve data to parameters provided by MeltWin while offering additional features including global fitting of data, auto-baseline generation, and two-state melting analysis. MeltR should be a useful tool for analyzing the next generation of DNA, RNA, and nonnucleic acid macromolecular melting data.

Estimation of Lewis Blood Group Status by Fluorescence Melting Curve Analysis in Simultaneous Genotyping of c.385A>T and Fusion Gene in FUT2 and c.59T>G and c.314C>T in FUT3

Lewis blood group status is determined by two fucosyltransferase activities: those of FUT2-encoded fucosyltransferase (Se enzyme) and FUT3-encoded fucosyltransferase (Le enzyme). In Japanese populations, c.385A>T in FUT2 and a fusion gene between FUT2 and its pseudogene SEC1P are the cause of most Se enzyme-deficient alleles (Sew and sefus), and c.59T>G and c.314C>T in FUT3 are tag SNPs for almost all nonfunctional FUT3 alleles (le59, le59,508, le59,1067, and le202,314). In this study, we first conducted a single-probe fluorescence melting curve analysis (FMCA) to determine c.385A>T and sefus using a pair of primers that collectively amplify FUT2, sefus, and SEC1P. Then, to estimate Lewis blood group status, a triplex FMCA was performed with a c.385A>T and sefus assay system by adding primers and probes to detect c.59T>G and c.314C>T in FUT3. We also validated these methods by analyzing the genotypes of 96 selected Japanese people whose FUT2 and FUT3 genotypes were already determined. The single-probe FMCA was able to identify six genotype combinations: 385A/A, 385T/T, sefus/sefus, 385A/T, 385A/sefus, and 385T/sefus. In addition, the triplex FMCA successfully identified both FUT2 and FUT3 genotypes, although the resolutions of the analysis of c.385A>T and sefus were somewhat reduced compared to that of the analysis of FUT2 alone. The estimation of the secretor status and Lewis blood group status using the form of FMCA used in this study may be useful for large-scale association studies in Japanese populations.

DNA concentration by solid phase reversible immobilization improves its yield and purity, and detection time of E. coli O157:H7 in foods by high resolution melt curve qPCR

For molecular detection of foodborne pathogens to be effective, an efficient technique for bacterial DNA concentration is required. Solid phase reversible immobilization (SPRI) paramagnetic beads can concentrate DNA from foods containing low amounts of bacteria because of their highly specific DNA-binding capacity. This study aimed to develop an upstream bacterial DNA concentration step using SPRI beads and compare its efficacy with immunomagnetic separation (IMS) for the detection of Shiga toxin producing E. coli (STEC) O157:H7 in food samples via a multiplex PCR assay. Food samples were inoculated with STEC O157:H7 at concentrations of 10, 100 and 1000 CFU/mL. The two different concentration methods were compared based on 1) DNA yield 2) DNA purity and 3) detection limit and time of the target pathogen using a high-resolution melt cure multiplex real-time PCR assay for the detection of the major virulence genes, eaeA, stx1 and stx2, in E. coli O157:H7. DNA samples isolated and concentrated by SPRI showed better purity and yield compared to IMS cell concentration. A detection limit of 10 CFU was attained for the majority of inoculated food samples utilizing DNA samples concentrated using SPRI in just 4 h as opposed to 6 or more hours using IMS. SPRI paramagnetic beads in combination with multiplex HRM real-time PCR can be used to rapidly concentrate and detect low concentrations of E. coli O157:H7 in food samples that cannot be detected by conventional culture techniques.

Development of a Real-Time PCR Assay for Detection of Macrolide Resistance Mutations in Mycoplasma genitalium and Its Application for Epidemiological Surveillance in Russia.

A real-time PCR assay based on probe fluorescence quenching by a primer was developed and validated for detection of macrolide resistance (MR)-associated mutations in the 23S rRNA gene of Mycoplasma genitalium. The assay involves identification of any nucleotide substitutions at positions 2058, 2059, and 2611 of 23S rRNA (Escherichia coli numbering) by the probe-based melting curve analysis immediately after amplification and was capable of detecting target mutations in clinical specimens and spiked samples with 92% sensitivity and 100% specificity. We applied this new assay to assess the prevalence of MR-associated mutations in 949 nonduplicate urogenital samples positive for M. genitalium by routine diagnostic PCR, which were collected from symptomatic patients in five cities in the European part of Russia during the period 2009-2019. Forty-three (4.92%) samples revealed the presence of MR mutations, and no trend toward an increase in resistance prevalence was observed over the 10-year period of the study. The most commonly detected mutations were A2058G (26/43; 60.47%) and A2059G (13/43; 30.23%), while other mutations were rare: A2058T (3/43; 6.98%) and C2611T (1/43; 2.33%). The data obtained underline the need for regular epidemiological monitoring to ensure effective patient management, rational use of antibiotics, and prevention of further spread of resistance.

Investigation of bedaquiline resistance and genetic mutations in multi-drug resistant Mycobacterium tuberculosis clinical isolates in Chongqing, China

BackgroundTo investigate the prevalence and molecular characterization of bedaquiline resistance among MDR-TB isolates collected from Chongqing, China.MethodsA total of 205 MDR-TB isolates were collected from Chongqing Tuberculosis Control Institute between March 2019 and June 2020. The MICs of BDQ were determined by microplate alamarblue assay. All strains were genotyped by melting curve spoligotyping, and were subjected to WGS.ResultsAmong the 205 MDR isolates, the resistance rate of BDQ was 4.4% (9/205). The 55 (26.8%) were from male patients and 50 (24.4%) were new cases. Furthermore, 81 (39.5%) of these patients exhibited lung cavitation, 13 (6.3%) patients afflicted with diabetes mellitus, and 170 (82.9%) isolates belonged to Beijing family. However, the distribution of BDQ resistant isolates showed no significant difference among these characteristics. Of the 86 OFX resistant isolates, 8 isolates were XDR (9.3%, 8/86). Six BDQ resistant isolates (66.7%, 6/9) and two BDQ susceptible isolates (1.0%, 2/196) carried mutations in Rv0678. A total of 4 mutations types were identified in BDQ resistant isolates, including mutation in A152G (50%, 3/6), T56C (16.7%, 1/6), GA492 insertion (16.7%, 1/6), and A274 insertion (16.7%, 1/6). BDQ showed excellent activity against MDR-TB in Chongqing.ConclusionsBDQ showed excellent activity against MDR-TB in Chongqing. The resistance rate of BDQ was not related to demographic and clinical characteristics. Mutations in Rv0678 gene were the major mechanism to BDQ resistance, with A152G as the most common mutation type. WGS has a good popularize value and application prospect in the rapid detection of BDQ resistance.

Estimation of suitable upper-limits for temperature, in stability comparisons between solid phases at high pressures. Study cases: carbon, oxygen, and fluorine

For most solid materials the Quasi-Harmonic Approximation (QHA) is expected to stay valid in a wide range of temperatures that get extended as we increase pressure, reaching a limit that could vastly exceed the melting temperature of the material. Hence, it becomes relevant to develop additional criteria for constraining the maximum temperature under which it still makes sense to perform stability comparisons between crystalline phases, hopefully without paying the high computational price that is required to calculating the precise melting curve for each solid phase. In this work, we report that for crystalline systems in which QHA remains accurate at high temperature, an alternative and computationally inexpensive phenomenological approximation holds well for identifying the region in the pressure-temperature (P-T) space where melting is likely to occur, meaning, the Lindemann’s criteria. By quantifying how atoms deviate from their equilibrium positions upon increasing temperature in the solid phase, we were able to constrain their P-T region of stability to conditions that are located under a line that represents an 11% average deviation of the atoms with respect to their interatomic distances, therefore, providing an approximate but reliable lower limit for the melting line which is relatively easy to calculate, and also a very useful alternative when no accurate experimental data is available and calculations do not exist or are not conclusive.

A rapid, accurate, and low-cost method for detecting Mycobacterium tuberculosis and its drug-resistant genes in pulmonary tuberculosis: Applications of MassARRAY DNA mass spectrometry.

Mycobacterium tuberculosis (MTB) identification and drug resistance diagnosis are very important for treatment of drug-resistant tuberculosis (DR-TB). Therefore, high throughput, accurate and low-cost molecular detection techniques are urgently needed. This study aimed to evaluate the clinical application value of MassARRAY in tuberculosis diagnosis and drug resistance screening. The limit of detection (LOD) and clinical application value of MassARRAY were evaluated using reference strains and clinical isolates. MTB in bronchoalveolar lavage fluid (BALF) and sputum samples were detected using MassARRAY, quantitative real-time polymerase chain reaction (qPCR) and MGIT960 liquid culture (culture). Using culture as the standard, the efficacy of MassARRAY and qPCR for the detection of TB was analyzed. Mutation of drug resistance genes in MTB clinical isolates was tested using MassARRAY, high-resolution melting curve (HRM), and Sanger sequencing. Using sequencing as the standard, the efficacy of MassARRAY, and HRM for the detection of each drug resistance site of MTB was analyzed. Simultaneously, the mutation of drug resistance genes by the MassARRAY method was compared with the results of drug susceptibility testing (DST), and the genotype-phenotype relationship was analyzed. The ability of MassARRAY to discriminate mixed infections was detected using mixtures of standard strains (M. tuberculosis H37Rv) and drug-resistant clinical isolates and mixtures of wild-type and mutant plasmids. In MassARRAY, 20 related gene mutations could be detected by two PCR systems. All genes could be accurately detected when the bacterial load was 104 CFU/mL. When the load of wild-type and drug-resistant MTB mixture was 105 CFU/mL (respectively reached 104 CFU/mL), variants and wild-type genes could be detected simultaneously. The sensitivity of MassARRAY (96.9%) for identification was higher than that of qPCR (87.5%) (p < 0.001). The sensitivity and specificity of MassARRAY for all drug resistance gene mutations were 100.0%, with higher accuracy and consistency than HRM (sensitivity = 89.3% and specificity = 96.9%, p = 0.001). Analyzing the relationship between MassARRAY genotype and DST phenotype, the accuracy of katG_315, rpoB_531, rpsL_43, rpsL_88, and rrs_513 sites was 100.0%, and embB_306 and rpoB_526 were inconsistent with the DST results when the base changes were different. MassARRAY can obtain base mutation information and identify heteroresistance infections simultaneously when the mutant proportion was at least 5-25%. It has good application prospects in the diagnosis of DR-TB with high throughput, accurate and low-cost.

Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients

A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity.

Multiplex polymerase spiral reaction for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are common food-borne pahogens that cause food poisoning in humans. In this study, we developed a method for the simultaneous determination of S. typhimurium and S. aureus based on multiplex polymerase spiral reaction (m-PSR) and melting curve analysis. Two pairs of primers were designed specifically to target the conserved invA gene of S. typhimurium and nuc gene of S. aureus, and the nucleic acid amplification reaction was achieved under isothermal conditions in the same reaction tube for 40 min at 61 °C, melting curve analysis of the amplification product was carried out. The distinct mean melting temperature allowed simultaneous differentiation of the two target bacteria in the m-PSR assay. The limit of detection of S. typhimurium and S. aureus that could be detected simultaneously was 4.1 × 10−4 ng genomic DNA and 2 × 101 CFU/mL pure bacterial culture. Based on this method, analysis of artificially contaminated samples showed excellent sensitivity and specificity consistent with those of pure bacterial cultures. This method is rapid, simultaneous and promises to be a useful tool for the detection of food-borne pathogens in the food industry.

RT-qPCR genotyping assays for differentiating Rift Valley fever phlebovirus strains

Rift Valley fever phlebovirus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen. Real time RT-qPCR genotyping (GT) assays were developed to differentiate between two RVFV wild-type strains (128B-15 and SA01–1322) and a vaccine strain (MP-12). The GT assay uses a one-step RT-qPCR mix, with two different RVFV strain-specific primers (either forward or reverse) with long or short G/C tags and a common primer (either forward or reverse) for each of the 3 genomic segments. The GT assay produces PCR amplicons with unique melting temperatures that are resolved in a post PCR melt curve analysis for strain identification. Furthermore, a strain specific RT-qPCR (SS-PCR) assay was developed to allow for specific detection of low titer RVFV strains in mixed RVFV samples. Our data shows that the GT assays are capable of differentiating L, M, and S segments of RVFV strains 128B-15 versus MP-12, and 128B-15 versus SA01–1322. The SS-PCR assay results revealed that it can specifically amplify and detect a low titer MP-12 strain in mixed RVFV samples. Overall, these two novel assays are useful as screening tools for determining reassortment of the segmented RVFV genome during co-infections, and could be adapted and applied for other segmented pathogens of interest.

Association of Androgen-Receptor Gene Mutations with the Copy Number of Androgen-Receptor Silk Protein A Complex and Glutathione-S-Transferases T1 and M1 in Prostate Cancer Patients.

The purpose of our work was to explore the association of mutations in the androgen receptor gene and copy numbers of the androgen-receptor silk protein A complex with glutathione-S-transferases T1 and M1 in prostate cancer patients. Eighty-five patients with PC and 85 healthy controls were included in the study. Fasting peripheral venous blood was collected, whole blood genomic DNA was extracted, and AR gene-receptor genotype was detected by a high-resolution melting curve analysis detection technology. Expression levels of androgen receptor (AR) and filamin protein A (FlnA) were detected by Western blotting. RT-PCR was used to detect the copy number of T1 and M1 glutathione-S-transferases. The wild-type androgen receptor gene rs5918762 is of TT type. The frequencies of CC and TC genes in the prostate cancer group were significantly higher than those in the normal control group (P < 0.05). Compared with TT-type PC patients, PC patients with TC-type and CC-type had higher expression levels of sex hormone receptor silk protein A complex and higher copy numbers of GSTT1 and GSTM1 (P < 0.05). Androgen-receptor gene mutation (T ⟶ C) was significantly positively correlated with the expression level of androgen-receptor silk protein A complex and the copy number of GSTT1 and GSTM1. Androgen-receptor gene polymorphisms were significantly associated with expression levels of androgen receptor complex A and silk proteins, and copy numbers of T1 and M1 glutathione-S-transferases. A combination of four factors can be used to identify prostate cancer susceptibility and disease progression.

A comprehensive survey of permethrin resistance in human head louse populations from northwest Iran: ex vivo and molecular monitoring of knockdown resistance alleles

BackgroundHead louse infestation is an important public health problem, and expanding resistance to permethrin is a major challenge to its control. The mapping and detection of pyrethroid resistance are essential to the development of appropriate treatments and ensure the effectiveness of current measures. The aim of this study was to present the phenotypic and genotypic basis of permethrin resistance and identify knockdown resistance (kdr) mutations in head louse populations in northwestern Iran.MethodsAdult head lice were collected from 1059 infested girls in Ardebil, East Azerbaijan, West Azerbaijan and Zanjan Provinces, northwestern Iran. The toxicity of permethrin and the possible synergistic effect of piperonyl butoxide (PBO) on this toxicity were assessed using bioassays. Fragments of voltage-sensitive sodium channels (vssc) and cytochrome b (cytb) genes were amplified and analyzed for the detection of knockdown resistance (kdr) mutations and mitochondrial groups. Moreover, genotypes of the two hot spot regions of the vssc gene were determined by melting curve analysis of amplicons.ResultsA total of 1450 adult head lice were collected during 2016–2021. Live lice were exposed to a dose of 1% permethrin for 12 h, and the median lethal time (LT50) and time to achieve 90% mortality (LT90) were determined to be 6 and 14.8 h, respectively. Topical application of 2 and 16 ng permethrin per louse resulted in 25% and 42.11% mortality, respectively. Pre-exposure of samples to 3% piperonyl butoxide had no synergistic effect on the effects of permethrin. Analysis of the 774-bp vssc gene fragment showed the presence of the M815I, T917I and L920F mutations, wild-type and T917I mutation, in 91.6%, 4.2% and 4.2% of samples, respectively. Investigation of the mitochondrial cytb gene demonstrated the predominance of clade B. The frequency of domain II segment 4 (S4)-S5 kdr genotypes in mitochondrial groups was identical, and heterozygotes were present in 93.5% of samples. A significant difference was detected in the frequency of domain IIS1-S3 kdr genotypes, and the frequency of resistant alleles and heterozygotes was higher in clade B than in clade A.ConclusionsThe presence of kdr mutations in the vssc gene and the non-synergist effect of PBO indicate that pyrethroid target site insensitivity is the main resistance mechanism. This phenomenon and the high frequency of resistant alleles necessitate that new pediculosis management programs be developed. Further studies need to be conducted to identify all factors contributing this resistance and to develop alternative pediculicides.Graphical