Evaluation of single-cell sorting accuracy using antibody-derived tag-based qPCR.

Single-cell sorting (index sorting) is a widely used method to isolate one cell at a time using fluorescence-activated cell sorting (FACS) for downstream applications such as single-cell sequencing or single-cell expansion. Despite widespread use, few assays are available to evaluate the proteomic features of the sorted single cell and further confirm the accuracy of single-cell sorting. With this caveat, we developed a novel assay to confirm the protein expression of sorted single cells by co-staining cells with the same marker using both antibody-derived tags (ADTs) and fluorescent antibodies. After single-cell sorting, we amplified the oligo of the ADT reagent as a surrogate signal for the protein expression using multiplex TaqMan™ qPCR on sorted cells. This assay is not only useful for confirming the identity of a sorted single cell but also an efficient method to profile proteomic features at the single-cell level. Finally, we applied this assay to characterize protein expression on whole cell lysate. Because of the sensitivity of the TaqMan™ qPCR, we can detect protein expression from a small number of cells. In summary, the ADT-based qPCR assay developed here can be utilized to confirm single-cell sorting accuracy and characterizing protein expression on both single cells and whole cell lysate.

Antibiotic-induced microbiome depletion promotes intestinal colonization by Campylobacter jejuni in mice.

BACKGROUND: To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion. RESULTS: Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes. CONCLUSIONS: Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1 ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG + GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

Development of TaqMan-based real-time PCR based on ψ gene for quantitative detection of CAR-T cells.

Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/µL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.

Characteristics of the a sequence of the duck Plague virus genome and specific cleavage of the viral genome based on the a sequence.

During the replication process, the herpesvirus genome forms the head-to-tail linked concatemeric genome, which is then cleaved and packaged into the capsid. The cleavage and packing process is carried out by the terminase complex, which specifically recognizes and cleaves the concatemeric genome. This process is governed by a cis-acting sequence in the genome, named the a sequence. The a sequence and genome cleavage have been described in some herpesviruses, but it remains unclear in duck plague virus. In this study, we analysed the location, composition, and conservation of a sequence in the duck plague virus genome. The structure of the DPV genome has an a sequence of (DR4)m-(DR2)n-pac1-S termini (32 bp)-L termini (32 bp)-pac2, and the length is 841 bp. Direct repeat (DR) sequences are conserved in different DPV strains, but the number of DR copies is inconsistent. Additionally, the typical DR1 sequence was not found in the DPV a sequence. The Pac1 and pac2 motifs are relatively conserved between DPV and other herpesviruses. Cleavage of the DPV concatemeric genome was detected, and the results showed that the DPV genome can form a concatemer and is cleaved into a monomer at a specific site. We also established a sensitive method, TaqMan dual qRT‒PCR, to analyse genome cleavage. The ratio of concatemer to total viral genome was decreased during the replication process. These results will be critical for understanding the process of DPV genome cleavage, and the application of TaqMan dual qRT‒PCR will greatly facilitate more in-depth research.

Development of a diagnostic variable number tandem repeat marker and dual TaqMan genotyping assay to distinguish Lophophora species.

The Lophophora genus of the Cactaceae family includes Lophophora diffusa and Lophophora williamsii, which has traditionally been used as a natural analgesic; however, its use is now under strict regulation worldwide as it contains mescaline, a unique psychotropic agent. Recently, non-medical and illegal distribution and abuse of L. williamsii have increased worldwide; thus, effective species identification methods are urgently needed. Here, we identified a new variable number tandem repeat (VNTR) marker in the trnL intron region to identify and characterize species in forensic analyses. The VNTR marker has a unique structure of tandem repeats, each with 13 nucleotides; one repeat unit was found in L. williamsii and two in L. diffusa. Phylogenetic and length polymorphism analyses confirmed that this novel VNTR marker could distinguish between Lophophora species. Furthermore, our newly developed TaqMan genotyping assay utilizes two probes; the color and position of dots on the discrimination plot differ according to the tandem repeat count within the VNTR marker. The limits of detection of the assay were 0.000063 ng (LW-VNTR probe-1) and 0.000066 ng (LW-VNTR probe-2), indicating high sensitivity. Moreover, when crime scene samples of 16 presumed L. williamsii species were analyzed, the results coincided with those of gas chromatography-mass spectrometry, confirming the applicability of our marker for Lophophora species identification. Thus, the tandem repeats within the trnL intron region can be exploited as a VNTR marker to identify L. williamsii and L. diffusa. Our dual TaqMan genotyping assay based on a novel marker demonstrates potential for forensic applications.

Highly Sensitive Dual-Probe Fluorescence Assay Based on an Aptamer for the Detection of Sulfasalazine.

Sulfadiazine (SD) is extensively utilized in agriculture, aquaculture, poultry, medical, and other industries. Its residues pose a threat to human health by entering the food chain and can also be released into the environment through animal feces and urine, leading to ecotoxicological pollution. Consequently, there is an urgent need to establish an efficient method for detecting SD residues in the environment. In this study, a novel two-probe fluorescence assay for determining SD in the environment, based on magnetic separation and real-time quantitative PCR-TaqMan probe technology, was successfully developed. In the experiment, SD served as the target substance, and an aptamer (Apt) with high affinity for SD was synthesized. Additionally, a non-fully complementary chain (Cdna) with favorable hybridization properties with the aptamer was designed and synthesized to create a magnetic probe of magnetic beads@Apt@Cdna. When SD was introduced, Apt specifically bound to SD with a hairpin structure and was released from the magnetic probe, allowing SD detection via the PCR-TaqMan method. Factors affecting the determination accuracy of this assay system, such as Apt concentration, SD standard solution pH, and incubation time, were optimized. Under the optimized conditions, the assay demonstrated high sensitivity for SD, with a detection limit of 2.34 × 10-5 ng/mL. Finally, the method was applied to detect SD in water samples from the Jialu River Basin in Zhengzhou City, yielding spiked recoveries of 88.82-117.05%. The results indicated that the detection system is a highly sensitive and specific method for determining SD residues in environmental water samples, showcasing its potential application in SD detection.

Association of circulating hsa-miRNAs with sarcopenia: the SarcoPhAge study.

OBJECTIVE: To identify a microRNA signature associated to sarcopenia in community-dwelling older adults form the SarcoPhAge cohort. METHODS: In a screening phase by next generation sequencing (NGS), we compared the hsa-miRome expression of 18 subjects with sarcopenia (79.6 ± 6.8 years, 9 men) and 19 healthy subjects without sarcopenia (77.1 ± 6 years, 9 men) at baseline. Thereafter, we have selected eight candidate hsa-miRNAs according to the NGS results and after a critical assessment of previous literature. In a validation phase and by real-time qPCR, we then analyzed the expression levels of these 8 hsa-miRNAs at baseline selecting 92 healthy subjects (74.2 ± 10 years) and 92 subjects with sarcopenia (75.3 ± 6.8 years). For both steps, the groups were matched for age and sex. RESULTS: In the validation phase, serum has-miRNA-133a-3p and has-miRNA-200a-3p were significantly decreased in the group with sarcopenia vs controls [RQ: relative quantification; median (interquartile range)]: -0.16 (-1.26/+0.90) vs +0.34 (-0.73/+1.33) (p < 0.01) and -0.26 (-1.07/+0.68) vs +0.27 (-0.55/+1.10) (p < 0.01) respectively. Has-miRNA-744-5p was decreased and has-miRNA-151a-3p was increased in the group with sarcopenia vs controls, but this barely reached significance: +0.16 (-1.34/+0.79) vs +0.44 (-0.31/+1.00) (p = 0.050) and +0.35 (-0.22/+0.90) vs +0.03 (-0.68/+0.75) (p = 0.054). CONCLUSION: In subjects with sarcopenia, serum hsa-miRNA-133a-3p and hsa-miRNA-200a-3p expression were downregulated, consistent with their potential targets inhibiting muscle cells proliferation and differentiation.

Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

TaqMan Real-Time PCR Assay for Specific Detection and Differentiation of Xanthomonas translucens pv. undulosa from Other Pathovars Targeting a Recombination Mediator Gene, recF.

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seedborne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene (recF). Analysis of the complete recF (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa, differentiating it from the other pathovars; recF-based sequence homology values among the 11 pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of recF to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa. The specificity of the assay was validated using 67 bacterial and fungal/oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of 11 pv. undulosa/secalis strains. The 56 strains of other X. translucens pathovars (n = 39) and non-Xanthomonas spp. (n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers.

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Identification and evaluation of potential inhibitor molecules against TcyA from Candidatus Liberibacter asiaticus.

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

Application of droplet digital PCR combined with TaqMan real-time PCR in Dombrock blood group genotyping in Northwest China.

BACKGROUND AND OBJECTIVES: The Dombrock blood group system is based on the DO gene. DO*A and DO*B antigens are the result of a single-nucleotide polymorphism (SNP) on this gene. The introduction of Do antigens through blood transfusion or other invasive factors like infection may result in the production of Do antibodies, which may cause serious haemolytic transfusion reactions. In this study, TaqMan real-time PCR and droplet digital PCR were used to detect rare DO*A allele, guide the search for rare DO*A allele donors, and calculate DO alleles frequencies in mixed populations in Northwest China. MATERIALS AND METHODS: In this study, the highly sensitive and accurate TaqMan real-time polymerase chain reaction (PCR) method was used to detect and screen DO genotype SNPs in combination with droplet digital PCR. We also searched for rare DO*A allele donors and calculated the frequencies of DO alleles in mixed populations. RESULTS: A total of 1202 donor DNA samples were collected from Northwest China, of which 202 were used to detect DO allele SNPs using TaqMan real-time PCR. The rare DO*A allele was detected in the other 1000 blood donors by droplet digital PCR, and gene frequencies were inferred from dual channel droplet digital PCR data. Among 1202 donors from Northwest China, the allele frequencies of DO*A and DO*B were 0.1128 and 0.8872, respectively. CONCLUSION: The sequencing results confirmed that this new way of detecting DO alleles by droplet digital PCR with specific probes can detect rare DO*A allele to predict the presence of the rare antigen Doa and infer DO allele frequencies. This method is highly sensitive and specific.

In vitro production and multiplex quantification of 2',3'-cyclic phosphate-containing 5'-tRNA half molecules.

RNA cleavages by many ribonucleases generate RNA molecules that contain a 2',3'-cyclic phosphate (cP) at their 3'-termini, and many cP-containing RNAs (cP-RNAs) are expressed as functional molecules in cells and tissues. 5'-tRNA half molecules are representative examples of functional cP-RNAs, playing important roles in various biological processes. We here show in vitro production of cP-containing 5'-tRNA half molecules that is able to prepare abundant synthetic cP-RNAs enough for functional analyses. Furthermore, we report a multiplex TaqMan RT-qPCR method which can simultaneously quantify multiple cP-containing 5'-tRNA half species. The method enabled us to efficiently quantify 5'-tRNA halves using samples with limited amounts, such as human plasma samples, revealing drastic enhancement of 5'-tRNA half levels at approximately 1,000-fold in patients infected with Mycobacterium tuberculosis. These in vitro production and multiplex quantification methods can be applied to any cP-RNAs, and they provide cost-effective, in-house techniques to accelerate expressional and functional characterizations of 5'-tRNA halves and other cP-RNAs.

Study the association of microRNA polymorphisms (miR-146a, miR-4513) with the risk of coronary heart diseases in Egyptian population.

Coronary heart disease (CHD) is the most prevalent cause of cardiovascular mortality in the world. It is well established that microRNAs (miRNAs) and their variants have an essential role in regulating the development of cardiovascular physiology, thus impacting the pathophysiology of heart diseases. This study was designed to determine the possible association of miRNA polymorphisms (miRNA-146a rs2910164C/G and miR-4513 rs2168518G/A) with susceptibility to CHD in Egyptian patients and their correlation with different biochemical parameters. The study comprised 300 participants, including 200 unrelated patients with CHD and 100 healthy controls. Anthropometric and blood biochemical parameters were measured as well genetic analysis for rs2910164C/G and rs2168518G/A polymorphisms were performed for all subjects using TaqMan real-time PCR assay. Our results revealed that the biomedical parameters have a significant correlation between CHD patients and healthy controls with a p < 0.05. Analyses of genotype distribution for (rs2910164 and rs2168518) revealed a significant association with CHD [odd ratio = 4.54, confidence interval (CI 95%) = (2.41-8.53)] and [odd ratio = 0.88, (CI 95%) = (0.83-0.92)], respectively. Furthermore, a statistically significant difference was detected between lipid profile levels and both rs2910164 and rs2168518 polymorphisms. The present study's findings indicated that the selected polymorphisms, miR-146a rs2910164 and miR-4513 rs2168518 could represent a useful biomarker for susceptibility to CHD in the Egyptian population. These genetic characteristics and personal habits and environmental factors may contribute to the development of CHD.

Two species-specific TaqMan-based quantitative polymerase chain reaction assays for the detection in soil of Paenibacillus polymyxa inocula.

AIMS: The increasingly widespread use of beneficial microbial inocula in agriculture gives rise to two primary needs: i) the assessment of the environmental risk, i.e. their impact on local soil microbiome and soil properties; ii) being able to track them and monitor their persistence and fate to both optimize their formulation and application method. In previous years, PCR-based methods have detected bacterial or fungal bioinoculant at the species or strain level. However, the selective detection, quantification, and monitoring of target microbial species in a complex ecosystem such as soil require that the tests possess high specificity and sensitivity. METHODS AND RESULTS: The work proposes a quantitative real-time PCR detection method using TaqMan chemistry, showing high specificity and sensitivity for the Paenibacillus polymyxa K16 strain. The primer and probe sets were designed using the polymyxin gene cluster targeting pmxC and pmxE sequences. Validation tests showed that these assays allowed a discriminant and specific detection of P. polymyxa K16 in soil. CONCLUSION: The TaqMan-assay developed could thus ensure the necessary level of discrimination required by commercial and regulatory purposes to detect and monitor the bioinoculant in soil.

Cross-Resistance of Succinate Dehydrogenase Inhibitors (SDHI) in Botrytis cinerea and Development of Molecular Diagnostic Tools for SDHI Resistance Detection.

Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs (pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen) with commonly used SDHIs boscalid and fluopyram. Different mutations were detected in the sdhB gene in B. cinerea collected from Michigan grapes, and their frequency and EC50 value were determined. Among 216 B. cinerea boscalid-resistant isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I, at frequencies of 82.6, 4.3, 11.5, 0.4, and 5.3%, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We classified the resistance profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants identified in this study. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea. We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤10 pg and in a single assay was capable of monitoring two amino acid positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control.

Monitoring Spore Dispersal and Early Infections of Diplocarpon coronariae Causing Apple Blotch Using Spore Traps and a New qPCR Method.

Apple blotch (AB) is a major disease of apple in Asia and recently emerged in Europe and the United States. It is caused by the fungus Diplocarpon coronariae (formerly Marssonina coronaria; teleomorph: Diplocarpon mali) and leads to severe defoliation of apple trees in late summer, resulting in reduced yield and fruit quality. To develop effective disease management strategies, a sound knowledge of the pathogen's biology is crucial. Data on the early phase of disease development are scarce: No data on spore dispersal in Europe are available. We developed a highly sensitive TaqMan qPCR method to quantify D. coronariae conidia in spore trap samples. We monitored temporal and spatial dispersal of conidia of D. coronariae and the progress of AB in spring and early summer in an extensively managed apple orchard in Switzerland in 2019 and 2020. Our results show that D. coronariae overwinters in leaf litter, and spore dispersal and primary infections occur in late April and early May. We provide the first results describing early-season dispersal of conidia of D. coronariae, which, combined with the observed disease progress, helps to understand the disease dynamics and will be a basis for improved disease forecast models. Using the new qPCR method, we detected D. coronariae in buds, on bark, and on fruit mummies, suggesting that several apple tissues might serve as overwintering habitats for the fungus, in addition to fallen leaves. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Etiology of acute febrile illness in the peruvian amazon as determined by modular formatted quantitative PCR: a protocol for RIVERA, a health facility-based case-control study.

BACKGROUND: The study of the etiology of acute febrile illness (AFI) has historically been designed as a prevalence of pathogens detected from a case series. This strategy has an inherent unrealistic assumption that all pathogen detection allows for causal attribution, despite known asymptomatic carriage of the principal causes of acute febrile illness in most low- and middle-income countries (LMICs). We designed a semi-quantitative PCR in a modular format to detect bloodborne agents of acute febrile illness that encompassed common etiologies of AFI in the region, etiologies of recent epidemics, etiologies that require an immediate public health response and additional pathogens of unknown endemicity. We then designed a study that would delineate background levels of transmission in the community in the absence of symptoms to provide corrected estimates of attribution for the principal determinants of AFI. METHODS: A case-control study of acute febrile illness in patients ten years or older seeking health care in Iquitos, Loreto, Peru, was planned. Upon enrollment, we will obtain blood, saliva, and mid-turbinate nasal swabs at enrollment with a follow-up visit on day 21-28 following enrollment to attain vital status and convalescent saliva and blood samples, as well as a questionnaire including clinical, socio-demographic, occupational, travel, and animal contact information for each participant. Whole blood samples are to be simultaneously tested for 32 pathogens using TaqMan array cards. Mid-turbinate samples will be tested for SARS-CoV-2, Influenza A and Influenza B. Conditional logistic regression models will be fitted treating case/control status as the outcome and with pathogen-specific sample positivity as predictors to attain estimates of attributable pathogen fractions for AFI. DISCUSSION: The modular PCR platforms will allow for reporting of all primary results of respiratory samples within 72 h and blood samples within one week, allowing for results to influence local medical practice and enable timely public health responses. The inclusion of controls will allow for a more accurate estimate of the importance of specific prevalent pathogens as a cause of acute illness. STUDY REGISTRATION: Project 1791, Registro de Proyectos de Investigación en Salud Pública (PRISA), Instituto Nacional de Salud, Perú.