PCR-based Method For Detection Research Articles

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples

Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.

Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations.

The term new genomic techniques (NGTs) is an umbrella term used to describe a variety of techniques that can alter the genetic material of an organism and that have emerged or have been developed since 2001, when the existing genetically modified organism (GMO) legislation was adopted. The analytical framework used to detect GMOs in Europe is an established single harmonized procedure that is mandatory for the authorization of GM food and feed, thus generating a reliable, transparent, and effective labeling scheme for GMO products. However, NGT products can challenge the implementation and enforcement of the current regulatory system in the EU, relating in particular to the detection of NGT products that contain no foreign genetic material. Consequently, the current detection methods might fail to meet the minimum performance requirements. Although existing detection methods may be able to detect and quantify even small alterations in the genome, this does not necessarily confirm the distinction between products resulting from NGTs subject to the GMO legislation and other products. Therefore, this study provides a stepwise approach for the in silico prediction of PCR systems' specificity by testing a bioinformatics pipeline for amplicon and primer set searches in current genomic databases. In addition, it also empirically tested the PCR system evaluated during the in silico analysis. Two mutant genotypes produced by CRISPR-Cas9 in Arabidopsis thaliana were used as a case study. Overall, our results demonstrate that the single PCR system developed for identifying a nucleotide insertion in the grf1-3 genotype has multiple matches in the databases, which do not enable the discrimination of this mutated event. Empirical assays further support this demonstration. In contrast, the second mutated genotype, grf8-61, which contains a -3 bp deletion, did not yield any matches in the sequence variant database. However, the primer sequences were not efficient during the empirical assay. Our approach represents a first step in decision making for analytical methods for NGT detection, identification, and quantification in light of the European labeling regulations.

Quantum-First Trial: FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD)-Specific Measurable Residual Disease (MRD) Clearance Assessed through Induction (IND) and Consolidation (CONS) Is Associated with Improved Overall Survival (OS) in Newly Diagnosed (nd) FLT3-ITD+ AML Patients (pts)

Background Detection of MRD by flow cytometry or gene fusion transcript quantitation increasingly is used to guide treatment decisions in AML. Molecular monitoring of recurrent gene mutations for MRD detection is an alternative strategy but, other than for nucleophosmin 1 ( NPM1) gene quantitation, remains controversial. FLT3-ITD is among the most common recurrent mutations in AML and confers a worse prognosis vs FLT3-wild-type and FLT3-TKD+ AML. Routine use of FLT3-ITD detection in remission as a predictor of relapse risk or OS has been limited by the low sensitivity of both conventional PCR-based detection methods and broad NGS platforms in clinical practice settings. More recently, FLT3-ITD-specific PCR-NGS assays such as getITD (PMID: 31089248) show greater sensitivity and ease of interpretation that point to eventual routine clinical application. However, the clinical value of these measurements has not been evaluated prospectively in large scale randomized controlled trials of FLT3 inhibitors. The phase 3 QuANTUM-First study (NCT02668653) evaluated the novel, potent, and highly selective type II FLT3 inhibitor quizartinib (Quiz) in nd FLT3-ITD+ AML pts and demonstrated that Quiz added to intensive IND and CONS, ± transplant, followed by single-agent continuation (CONT) therapy (Tx) resulted in a significant improvement in OS (PMID: 37116523). We analyzed if FLT3-ITD-specific MRD in QuANTUM-First pts impacted the clinical outcome or the benefits provided by Quiz in nd FLT3-ITD+ AML pts. Methods Genomic DNA, isolated from bone marrow aspirates or peripheral blood from pts after achievement of remission after 1 or 2 courses of IND and at end of CONS (prior to transplant or CONT cycle 1 day 1 [C1D1] for transplant pts and prior to CONT C1D1 for non-transplant pts), was analyzed with a FLT3-ITD PCR-NGS assay specifically developed for this trial (PMID: 31722002). ITD mutations detected after IND were cross-validated against the ITD detected at enrollment for each patient. Using a custom bioinformatics program, ITD mutations were identified, and variant allele frequencies (VAFs) were calculated with a sensitivity of 10 −5. MRD was classified as undetectable (using the 0 cutoff) or detectable above or below a 10 −4 predefined cutoff (based on lower limit of quantification for the assay). Comparisons of complete response (CR), composite complete response (CRc=CR+CRi), and the rate of pts achieving CRc during IND with no MRD between arms were made using a stratified Cochran-Mantel-Haenszel test. Comparison of FLT3-ITD VAF during IND and during CONS between arms was made using Wilcoxon rank sum test. All P-values were not adjusted for multiplicity. Results In QuANTUM-First, 539 nd FLT3-ITD+ AML pts were randomized to Quiz (n=268) or placebo (PBO; n=271). Of the 539 randomized pts, 368 (68.3%) achieved CRc after 1 or 2 courses of IND, and MRD analysis was performed on 321 (87.2%) of these pts (162 pts in Quiz and 159 pts in PBO) using samples collected at the time of response assessment during IND, before further Tx. MRD was also assessed in 337 pts (172 pts in Quiz and 165 pts in PBO) by end of up to 4 cycles of CONS prior to CONT/maintenance Tx. Of these pts, 166 additionally received transplant (87 in Quiz, 79 in PBO). The % of pts in CRc at end of IND with FLT3-ITD MRD of <10 −4 was similar between study arms (25.4% Quiz vs 21.8% PBO, nominal P = 0.3430), however, a greater % of pts in CRc had undetectable MRD with Quiz than PBO (12.3% vs 7.0%, nominal P = 0.0403). Among pts with CRc at end of IND, the median best FLT3-ITD VAF by end of CONS was lower (0% versus 0.0017%; nominal P = 0.0006) with Quiz vs PBO (Fig 1) and, using the 0 cutoff at end of IND, a longer OS was observed with Quiz vs PBO, regardless of MRD status, with a greater effect with Quiz (HR, 0.789 in MRD− pts; HR, 0.749 in MRD+ pts; Fig 2). In MRD+ pts, the median OS was not reached with Quiz and 35.4 months with PBO (Fig 2). Performing this analysis with an MRD cutoff of 10 ‒4 yielded similar findings (HR, 0.696 in MRD− pts; HR, 0.800 in MRD+ pts). Additional data about MRD impact on pts ± transplant in this trial will be presented. Conclusions These findings demonstrate the potential prognostic utility of FLT3-ITD-specific MRD measurements in the clinical management of pts with FLT3-ITD+ AML. Our data suggest that long-term OS benefits conferred by Quiz in part derive from a deep and sustained reduction of the FLT3-ITD+ leukemia burden.

An isothermal CRISPR-based diagnostic assay for Neisseria gonorrhoeae and Chlamydia trachomatis detection.

The occurrence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) is increasing worldwide, particularly in low- and middle-income countries. In cases where most infections are asymptomatic and remain in a coinfection condition. It may cause missed detections or the severe subsequent symptoms. Accurate diagnosis and convenient screening are essential for disease control. However, molecular diagnostics for CT/NG predominantly rely on polymerase chain reaction (PCR)-based detection, which requires special devices and conditions. Here, we developed an isothermal CRISPR-based CT/NG dual-target detection system by pairing recombinase polymerase amplification (RPA) and CRISPR-Cas12a/13a detection. Multiplex RPA can amplify NG and CT simultaneously, and a single-pot reaction with the Cas12a/13a can be performed for further recognizing the amplified target, driving the cleavage of the corresponding fluorescence reporter separately; thus, the signal can be monitored in different channels. This CRISPR-based CT/NG detection system achieved analytical sensitivities of 10° copies/μL for both synthetic CT/NG dsDNA and exhibited no cross-reaction with other species. In tests on 88 clinical samples, our CRISPR-based assay showed excellent agreement with the commercial assay Roche Cobas 4800 (100% accuracy for NG, 94.32% accuracy for CT, and 97.73% accuracy for CT/NG coinfection) and showed higher positive percent agreement, negative percent agreement, and accuracy than those reported for TaqMan PCR in a previous study. In addition, the results of this CRISPR-based system could be acquired within 75 min. Our isothermal diagnostic method with promising performance provides more convenience than PCR-based methods for detection and screening.IMPORTANCEA method for Neisseria gonorrhoeae (NG)/Chlamydia trachomatis (CT) detection is developed using multiplex-recombinase polymerase amplification and Cas12a/Cas13a. This method can detect NG and CT simultaneously with high sensitivity and specificity. This method has great potential to be further developed into larger-scale screening and point-of-care testing (POCT).

B-239 Long-read Nanopore Sequencing is a Practicable Method of Targeted or Untargeted Microbial Contamination Detection

Abstract Background Sequencing-based methods for microbe detection are poised to replace both in vivo and PCR-based detection methods due to advantages in speed, sensitivity, specificity, and multiplexing. Here we describe pilot experiments to assess the practicability of nanopore sequencing for microbe detection. Methods Quantitative genomic Human Adenovirus 5 or Mycoplasma hominis DNA was spiked into remnant human serum and nucleic acids were extracted. 16S rRNA PCR amplification and barcoding was performed for Mycoplasma hominis identification. Sequencing was performed with a R9.4.1 flowcell on the MinION sequencer. Basecalling and genome alignment was performed with Guppy v.6.4.2. Results The performance of untargeted sequencing was evaluated by spiking quantitative Adenovirus DNA at 104 genome copies per microliter. Illustrative data from a single run is presented. Replicates yielded similar results. 1 520 991 reads between 31 bases and 970 kilobases were generated. 162 reads between 700 bases and 11.3 kilobases aligned to the Adenovirus genome (Image 1). To evaluate the performance of PCR amplified sequencing, we spiked Mycoplasma genomic DNA into serum or water. The entire 16S rRNA gene (1.5 kb) was PCR amplified and genomic DNA was spiked in at 104, 102, 10, and 0 (control) copies per microliter. A 10% signal: noise ratio cutoff yielded 100% specificity between spiked samples and negative controls. Conclusion Short read sequencing techniques are adequate for microbial detection but are capital intensive and prone to false positive results. We demonstrate that nanopore sequencing can generate long reads (>1.5 kb) with high-confidence microbe genome alignment. Our methods were sensitive to 104 and 10 copies per microliter utilizing untargeted and targeted methods, respectively. Further method optimization and validation will need to be performed, but these results demonstrate that nanopore sequencing is a practicable approach for microbe detection.

Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives

In this study levels and types of microbial contaminants were investigated in 88 different plant-based ingredients including many that are used to manufacture dairy alternatives. Studied ingredients encompassed samples of pulses (pea, faba bean, chickpea, and mung bean), cereals/pseudocereals (oat, rice, amaranth and quinoa) and drupes (coconut, almond and cashew). The microbial analysis included: i) total viable count (TVC), ii) total aerobic mesophilic spore count (TMS), iii) heat resistant aerobic thermophilic spore count (HRTS), iv) anaerobic sulfite reducing Clostridium spore count (SRCS), and v) Bacillus cereus spore count (BCES). Microorganisms isolated from the counting plates with the highest sample dilutions were identified using 16S rRNA and MALDI-TOF MS analyses.Many of the investigated ingredients showed a high proportion of spores as part of their total aerobic mesophilic counts. In 63 % of the samples, the difference between TVC and TMS counts was 1 Log10 unit or less. This was particularly the case for the majority of pea isolates and concentrates, faba bean isolates, oat kernels and flakes, and for single samples of chickpea isolate, almond, amaranth, rice, quinoa, and coconut flours. Concentrations of TVC ranged between <1.0 and 5.3 Log10 CFU/g in different samples, and TMS varied between <1.0 and 4.1 Log10 CFU/g. Levels of HTRS, BCES and SRCS were generally low, typically around or below the LOD of 1.0 Log10 CFU/g.In total, 845 individual bacterial colonies were isolated belonging to 33 different genera. Bacillus licheniformis and B. cereus group strains were most frequently detected among Bacillus isolates, and these species originated primarily from pea and oat samples. Geobacillus stearothermophilus was the main species encountered as part of the HRTS. Among the Clostridium isolates, Clostridum sporogenes/tepidum were predominant species, which were mostly found in pea and almond samples. Strains with potential to cause foodborne infection or intoxication were typed using the PCR-based method for toxin genes detection. In the B. cereus group, 9 % of isolates contained the ces gene, 28 % contained hbl, 42 % cytK, and 69 % were positive for the nhe gene. Absence of the boNT-A and -B genes was confirmed for all isolated C. sporogenes/tepidum strains. Nearly all (98 %) B. licheniformis isolates were positive for the lchAA gene.Insight into the occurrence of microbial contaminants in plant-based ingredients, combined with knowledge of their key inactivation and growth characteristics, can be used for the microbial risk assessment and effective design of plant-based food processing conditions and formulations to ensure food safety and prevent spoilage.

Identification and detection of microRNA kidney disease biomarkers in liquid biopsies.

MicroRNAs (miRNAs) are emerging rapidly as a novel class of biomarkers of major organ disorders, including kidney diseases. However, current PCR-based detection methods are not amenable to development for high-throughput, cost-effective miRNA biomarker quantification. MiRNA biomarkers show significant promise for diagnosis and prognosis of kidney diseases, including diabetic kidney disease, acute kidney injury, IgA nephropathy and delayed graft function following kidney transplantation. A variety of novel methods to detect miRNAs in liquid biopsies including urine, plasma and serum are being developed. As miRNAs are functional transcripts that regulate the expression of many protein coding genes, differences in miRNA profiles in disease also offer clues to underlying disease mechanisms. Recent findings highlight the potential of miRNAs as biomarkers to detect and predict progression of kidney diseases. Developing in parallel, novel methods for miRNA detection will facilitate the integration of these biomarkers into rapid routine clinical testing and existing care pathways. Validated kidney disease biomarkers also hold promise to identify novel therapeutic tools and targets. http://links.lww.com/CONH/A43.

Prevalence and Diversity of Blood Parasites (Plasmodium, Leucocytozoon and Trypanosoma) in Backyard Chickens (Gallus gallus domesticus) Raised in Southern Thailand.

Avian malaria and leucocytozoonosis can cause fatal diseases, whereas avian trypanosomiasis is reported to be harmless in chickens. Backyard chickens can be infected by several pathogens, including blood parasites, that may shed to industrial poultry production, with a consequently higher economic impact. This study aimed to investigate the presence of several blood parasites (Plasmodium, Leucocytozoon and Trypanosoma) in backyard chickens raised in Southern Thailand, using PCR-based detection and microscopic methods. From June 2021 to June 2022, 57 backyard chickens were sampled. Fresh thin blood smears were prepared from 11 individuals, and buffy coat smears were prepared from 55 of them. Both thin blood smears and buffy coat smears were used for microscopic analysis. Two nested PCR protocols that amplify a fragment of cytochrome b (cytb) and small subunit rRNA (SSU rRNA) genes were used to identify Haemosporida and Trypanosoma parasites, respectively. The number of positive samples was higher with the application of nested PCR than when buffy coat smears were used. Three new Plasmodium lineages (GALLUS47-49) and thirteen Leucocytozoon lineages (GALLUS50-62) were found. Trophozoites, meronts and gametocytes of Plasmodium gallinaceum (GALLUS01) were present in one thin blood smear. All thin blood smears revealed Leucocytozoon infections, but only three samples were a single infection. These three samples revealed the presence of fusiform host cell-parasite complexes, of which the morphological features resembled those of Leucocytozoon macleani (possible synonym is Leucocytozoon sabrazesi), while the cytb showed that this parasite is closely related to the lineage GALLUS06-07, described as Leucocytozoon schouteni. The Trypanosoma prevalence was 33.33%; it was present in only one of the thin blood smears, and it resembles Trypanosoma calmettei. This study showed the prevalence of a high diversity of Plasmodium (64.91%) and Leucocytozoon (89.47%) in Thai chickens. Both nested-PCR and buffy coat smear can be used as the diagnostic tool for the testing of Plasmodium, Leucocytozoon and Trypanosoma for parasitic control in backyard chickens and poultry farms. The information on the parasite species that can be found in chickens raised in Southern Thailand was also considered as the baseline information for further study.

Diagnostic performance of DNA probe-based and PCR-based molecular vaginitis testing.

Vaginitis is usually diagnosed empirically, microscopically, via cultures, or by molecular testing for the detection of bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), or Trichomonas vaginalis (TV). The DNA probe-based technique detects BV by identifying Gardnerella vaginalis, VVC by identifying Candida spp., while real-time PCR-based detection methods identify BV by algorithmic analysis of the absence or presence of known vaginal flora. We examined 8,878 total orders placed for DNA probe-based identification (ID) and 10,464 total orders placed for molecular panel ID. We found that PCR-based BV test positivity reduced from 30% to 23% compared with the population tested with DNA probe-based testing. We also found that PCR-based testing VVC positivity increased from 6.3% and 11.6% when compared with DNA probe-based testing. Bayesian generalized linear analysis estimated a lower mean proportion of positive tests for BV in PCR-based molecular panels than DNA probe testing suggesting an under-call of BV. The same models estimated a higher mean proportion of positive tests for molecular vaginal panels than DNA probe testing suggesting an increased detection of candidal vaginitis. In addition, the mean (SD) age for patients with Candida albicans was 40.5 (40.0-41.1) years. Patients with Candida glabrata (now N. glabrata) were 5.2-8.1 (mean 6.7) years older than patients with Candida albicans. Our retrospective data analysis found that BD Max MVP's ability to discriminate between vaginal candidiasis versus other yeast will help to implement CDC (Centers for Disease Control and Prevention)-recommended treatment options. We also believe that providers' inattention to non-albicans treatment could be an issue nationwide. IMPORTANCE Using retrospective data from U.S. Food and Drug Administration-approved/cleared molecular vaginal panels, molecular methods were found to have higher detection for Candida vaginitis and lower detection for bacterial vaginitis when compared to probe-based methods. In addition, the differentiation of Candida and non-Candida yeast has not reached the physician community as we observed noncompliance in recommended therapy. Furthermore, the pros and cons of migrating to molecular testing from conventional microscopy for identifying bacterial vaginitis and fungal vaginitis have been examined and reported in this paper. Interestingly, the mean (SD) age for patients with Candida albicans was 40.5 (40.0-41.1) years. Patients with N. glabrata were 5.2-8.1 (mean 6.7) years older than patients with Candida albicans.

Full-Length SMN Transcript in Extracellular Vesicles as Biomarker in Individuals with Spinal Muscular Atrophy Type 2 Treated with Nusinersen.

Three therapeutic strategies have radically changed the therapeutic scenario for spinal muscular atrophy (SMA). However, therapeutic response differs between individuals. There is a need to identify biomarkers to further assess therapeutic response and to better understand which variables determine the extent of response. We conducted a study using an optimized digital droplet PCR-based method for the ultra-sensitive detection of SMN transcript in serum EVs from SMA 2 individuals treated with nusinersen over 14 months. In parallel, we investigated levels of serum and CSF neurofilament heavy chain (pNF-H) in the same cohort. Expression of flSMN transcript in EVs of SMA 2 individuals prior to nusinersen was lower than in controls (0.40 vs 2.79 copies/ul; p < 0.05) and increased after 14 months of nusinersen (0.40 vs 1.11 copies/ul; p < 0.05). The increase in flSMN with nusinersen was significantly higher in younger individuals (p < 0.05). Serum pNF-h was higher in non-treated individuals with SMA 2 than in controls (230.72 vs 22.88 pg/ml; p < 0.05) and decreased with nusinersen (45.72 pg/ml at 6 months, 39.02 pg/ml at 14 months). CSF pNF-h in SMA 2 individuals also decreased with nusinersen (248.04 pg/ml prior to treatment, 197.10 pg/dl at 2 months, 104.43 pg/dl at 6 months, 131.03 pg/dl at 14 months). We identified an increase of flSMN transcript in serum EVs of SMA 2 individuals treated with nusinersen that was more pronounced in the younger individuals. Our results indicate that flSMN transcript expression in serum EVs is a possible biomarker in SMA to predict or monitor the response to treatment.

The application of a DNA nanobiosensor based on MXene/MWCNTs/PPY nanocomposite as a sensitive detection method of CEACAM5 for diagnosis of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is responsible for the high mortality rate related to gynecologic cancers. Due to the late-stage diagnosis, a 5-year survival rate is expected in only about 30% of patients. In this study, an electrochemical DNA biosensor based on MXene/MWCNTs/PPY nanocomposite was designed and introduced as an alternative method for Real-time PCR based detection methods of tumor markers. The biosensor specifically detected CEACAM5 as an ovarian cancer-related tumor marker in 26 real clinical samples. The statistical analysis results represented that the biosensor is able to differentiate between the case and control groups with 93.3 % sensitivity and high specificity. Moreover, the biosensor revealed a wide linear range of 5 × 10-11 − 5 × 10-7 M and good stability for up to 20 days. In conclusion, the fabricated biosensor provides a fast, low-cost, simple, and novel method for EOC detection.

Two-Dimensional (2D) materials in the detection of SARS-CoV-2

The SARS-CoV-2 pandemic has resulted in a devastating effect on human health in the last three years. While tremendous effort has been devoted to the development of effective treatment and vaccines against SARS-CoV-2 and controlling the spread of it, collective health challenges have been encountered along with the concurrent serious economic impacts. Since the beginning of the pandemic, various detection methods like PCR-based methods, isothermal nucleic acid amplification-based (INAA) methods, serological methods or antibody tests, and evaluation of X-ray chest results have been exploited to diagnose SARS-CoV-2. PCR-based detection methods in these are considered gold standards in the current stage despite their drawbacks, including being high-cost and time-consuming procedures. Furthermore, the results obtained from the PCR tests are susceptible to sample collection methods and time. When the sample is not collected properly, obtaining a false result may be likely. The use of specialized lab equipment and the need for trained people for the experiments pose additional challenges in PCR-based testing methods. Also, similar problems are observed in other molecular and serological methods. Therefore, biosensor technologies are becoming advantageous with their quick response, high specificity and precision, and low-cost characteristics for SARS-CoV-2 detection. In this paper, we critically review the advances in the development of sensors for the detection of SARS-CoV-2 using two-dimensional (2D) materials. Since 2D materials including graphene and graphene-related materials, transition metal carbides, carbonitrides, and nitrides (MXenes), and transition metal dichalcogenides (TMDs) play key roles in the development of novel and high-performance electrochemical (bio)sensors, this review pushes the sensor technologies against SARS-CoV-2 detection forward and highlights the current trends. First, the basics of SARS-CoV-2 detection are described. Then the structure and the physicochemical properties of the 2D materials are explained, which is followed by the development of SARS-CoV-2 sensors by exploiting the exceptional properties of the 2D materials. This critical review covers most of the published papers in detail from the beginning of the outbreak.

Triplex qPCR assay for Campylobacter jejuni and Campylobacter coli monitoring in wastewater

Campylobacter spp. is one of the most frequent pathogens of bacterial gastroenteritis recorded worldwide. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the two major disease-associated species, accounting for >95 % of infections, and thus have been selected for disease surveillance. Monitoring temporal variations in pathogen concentration and diversity excreted from community wastewater allows the early detection of outbreaks. Multiplex real-time/quantitative PCR (qPCR) enables multi-target quantification of pathogens in various types of samples including wastewater. Also, an internal amplification control (IAC) is required for each sample when adopting PCR-based methods for pathogen detection and quantification in wastewater to exclude the inhibition of the wastewater matrix. To achieve reliable quantification of C. jejuni and C. coli towards wastewater samples, this study developed and optimized a triplex qPCR assay by combining three qPCR primer-probe sets targeting Campylobacter jejuni subsp. jejuni, Campylobacter coli, and Campylobacter sputorum biovar sputorum (C. sputorum), respectively. This triplex qPCR assay not only can directly and simultaneously detect the concentration of C. jejuni and C. coli in wastewater but also can achieve the PCR inhibition control using C. sputorum primer-probe set. This is the first developed triplex qPCR assay with IAC for C. jejuni and C. coli, to be used in the wastewater-based epidemiology (WBE) applications. The optimized triplex qPCR assay enables the detection limit of the assay (ALOD100%) and wastewater (PLOD80%) as 10 gene copy/μL and 2 log10 cells/mL (2 gene copies/μL of extracted DNA), respectively. The application of this triplex qPCR to 52 real raw wastewater samples from 13 wastewater treatment plants demonstrated its potential as a high-throughput and economically viable tool for the long-term monitoring of C. jejuni and C. coli prevalence in communities and the surrounding environments. This study provided an accessible methodology and a solid foundation for WBE-based monitoring of Campylobacter spp. relevant diseases and paved the road for future WBE back-estimation of C. jejuni and C. coli prevalence.

Diagnosis of Soybean Diseases Caused by Fungal and Oomycete Pathogens: Existing Methods and New Developments

Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the genera Diaporthe, Sclerotinia, Colletotrichum, Fusarium, Cercospora, Septoria, Macrophomina, Phialophora, Rhizoctonia, Phakopsora, Phytophthora, and Pythium, causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.

Rapid molecular detection of CMY-2, and CTX-M group 1 and 9 variants via recombinase polymerase amplification.

Due to their prevalence worldwide, the β-lactamases CTX-M and plasmid-mediated CMY-2 are important antimicrobial resistance enzymes in a clinical setting. While culture- and PCR-based detection methods exist for these targets, they are time consuming and require specialist equipment and trained personnel to carry out. In this study, three rapid diagnostic single-plex and a prototype triplex assay were developed, using recombinase polymerase amplification with lateral flow detection (RPA-LF), and tested for their sensitivity and specificity using two isolate DNA panels (n = 90 and n = 120 isolates). In addition, the RPA-LF assays were also tested with a small number of faecal extract samples (n = 18). The RPA-LF assays were able to detect bla CXT-M-group-1, bla CTX-M-group-9 and bla CMY-2-type variants with high sensitivity (82.1%-100%) and specificity (100%) within a short turnaround time (15-20 min for amplification and detection). RPA-LF assays developed in this study have the potential to be used at or close to the point of care, as well as in low-resource settings, producing rapid results to support healthcare professionals in their treatment decisions.

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Molecular Characterization and Antimicrobial Susceptibility of Mycoplasma Species Isolated from Broilers and Breeder Chickens

Mycoplasmas are considered as important avian pathogens, which cause a great economical loss in the poultry industry. As it responsible for both respiratory disease and synovitis in poultry. The aim of this study was to isolate and identify Mycoplasma gallisepticum and Mycoplasma synoviae from chickens and determine the efficacy of different antibiotics. Additionally, detection of some substantial virulence genes. Two hundred chicken samples were collected and cultured onto specific PPLO medium .The isolates were characterized by polymerase chain reaction then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method. The results reported that 15 samples (7.5%) were positive by culturing with prevalent of Mycoplasma synoviae and Mycoplasma gallisepticum with 12 (80%) and three (20%). Following the 16S rRNA-based detection of Mycoplasma isolates, 12 Mycoplasma synoviae and three Mycoplasma synoviae isolates were identified by different PCR-based detection methods for various virulence genes. Three Mycoplasma gallisepticum isolates contain the mgc2 gene, and nine Mycoplasma synoviae isolates were positive for the ISR (intergenic spacer region) gene. All isolates had lowest MIC values for Tylvasolin with a range of (0.062-0.125μg/mL) followed by Lincomycin, Tiamulin, Tilmicosin and Tylosin with a range of (0.062-1 μg/mL). However, the Mycoplasma synoviae isolates displayed variance in MICs for Oxytetracycline with a range of 0.5 to 8 μg/mL, and Chlorotetracycline with a range of 2 to 8 μg/mL. Meanwhile, high MIC values for Enrofloxacin were detected in all isolates with MICs ranging from 8 to 32 μg/mL. Furthermore, the MIC method identified Tylvasolin, Lincomycin, Tiamulin, Tilmicosin and Tylosin as the antibiotics of choice for the treatment of MS infections. In conclusion, these data may help in prevention and control of Mycoplasma infection in poultry.

A Survey of Methicillin-Resistance Staphylococcus aureus Isolated from Patients Hospitalized in the ICU, NICU and Surgical Departments of Tehran University of Medical Sciences Hospitals

Background and Aim: Staphylococcus aureus is an important pathogen that causes various infections. The presence of some pathogenic factors in this bacterium helps the bacteria to survive in response to the immune system and antimicrobial treatments. Efforts to treat infections caused by these strains have resulted in resistance to most antibiotics, especially methicillin and vancomycin resistance, during recent years. Therefore, the consequences of infections caused by this bacterium can be serious. In this study we aimed to survey methicillin- resistance Staphylococcus aureus isolated from patients hospitalized in the ICU, NICU and surgical departments of Tehran University of Medical Sciences hospitals. Material and Method: 430 clinical samples of the patients were collected and identified as Staphylococcus aureus by phenotype and biotype tests. MRSA isolates were first screened by showing resistance to cefoxitin by disc diffusion method and finally confirmed by examining the mecA gene by PCR. The microbial resistance pattern of MRSA was also measured by disk diffusion method. Investigating the presence of a vancomycin resistance gene in isolated MRSA occurred by PCR. Results: Out of 430 clinical samples, 117 (27.20%) Staphylococcus aureus were isolated of which 42 (35.89%) were identified as MRSA. The most MRSA obtained from the ICU department with 25 cases (59.52%). The majority of patients infected with MRSA belonged to the age group of 40-65 years. The mecA gene was detected in all MRSA. The entire MRSA was 100% sensitive to vancomycin and linezolid, and 100% resistance to cloxacillin. Molecular detection of vanA gene in isolated MRSA showed 2 (4.76%) of them contained vanA gene. Conclusion: The high prevalence of MRSA and its antibiotic resistance pattern to vancomycin and other antibiotic families can lead to treatment failures, uncontrolled nosocomial infections, and mortality. Although the PCR-based detection method is superior to other conventional techniques, a combination of these methods can provide diagnostic accuracy.

Molecular Detection of Pentastiridius leporinus, the Main Vector of the Syndrome 'Basses Richesses' in Sugar Beet.

Monitoring of Pentastiridius leporinus (Hemiptera: Auchenorrhyncha: Cixiidae), representing the main vector of the syndrome 'basses richesses' (SBR) disease in sugar beet is based on morphological identification. However, two other cixiid species, Reptalus quinquecostatus and Hyalesthes obsoletus with similar external characters are known to appear in sugar beet fields and are challenging to be distinguished from P. leporinus. We present a PCR-based method for species-specific detection of both male and female P. leporinus, directly after sweep net collection or after up to 18 months long term storage on sticky traps. Two methods of DNA template preparation, based on a commercial extraction kit or on simple grinding in phosphate-buffered saline (PBS) were compared. The latter method was also established for eggs and all five nymphal instars of P. leporinus from a rearing. Furthermore, in silico primer analysis showed that all Auchenorrhyncha species including far related species reported from sugar beet fields can be differentiated from P. leporinus. This was PCR-confirmed for the most common Auchenorrhyncha species from different German sugar beet fields. Sequence analysis of the P. leporinus mitochondrial cytochrome oxidase I gene (COI) amplicon showed a close relationship to COI from P. beieri but separated from the Reptalus and Hyalesthes species which are grouped into the same family Cixiidae. We present a sensitive, cost- and time-saving PCR-based method for reliable and specific detection of eggs and all nymphal instars, as well as male and female P. leporinus, after different methods of planthopper collection and template DNA template preparation that can be used in large scale monitoring assays.

New PCR primers targeting the cytochrome b gene reveal diversity of Leucocytozoon lineages in an individual host.

Avian haemosporidian parasites have received considerable attention in ecology and evolution as a result of their wide distribution and ease of detection. However, conventional PCR-based detection methods may sometimes underestimate haemosporidian mixed infections, which are frequent in natural populations. This underestimation is due to differences in PCR sensitivity for detection of lineages within the mixed infections. Therefore, we designed new primers to amplify sequences that were not detected by the conventional primers and examined if our primers were useful for accurate detection of mixed infections. Blood samples were collected from 32 wild birds captured in Hokkaido, and 16 of these were positive for Leucocytozoon using the conventional primers, while 15 were positive using our primers. All positively amplified samples were sequenced, and we found that the conventional primers detected 16% (5/32) of multiple infections and none of them was a novel lineage, whereas our primers detected 44% (14/32) of multiple infections and ten of them were novel lineages. A phylogenetic analysis showed that the new primers can detect a wide range of Leucocytozoon lineages compared with that detected by the conventional primers. The results indicate that our primers are particularly suitable for revealing unique strains from multiple infections. Highly variable multiple infections in the same population of birds at the same location were found for the first time. We revealed a higher diversity of Leucocytozoon lineages in nature than expected, which would provide more information to better understand parasite diversity and host-vector interactions in wildlife.