Multiplex PCR Assay Research Articles

Toxigenic characterization, spoilage potential, and antimicrobial susceptibility of coagulase-positive Staphylococcus species isolated from Minas Frescal cheese

This study aimed to identify coagulase-positive staphylococci (CPS) species from 21 samples of clandestine Minas Frescal cheese, investigate the potential for deterioration in psychrotrophic and mesophilic conditions, verify the toxigenic potential of Staphylococcus aureus, and determine the antimicrobial susceptibility profile of toxigenic S. aureus. Species determination was performed based on the detection of β-hemolysis in 5% ovine blood agar; fermentation of mannitol, maltose, and trehalose sugars; and production of acetoin. After species determination, DNA extraction and analysis was performed for S. aureus colonies for genes encoding staphylococcal toxins (eta, etb, tst, sea, seb, sec, sed, and see) using 2 multiplex PCR assays. Isolates identified as toxigenic S. aureus were tested for antimicrobial susceptibility to tetracycline, erythromycin, clindamycin, gentamicin, ciprofloxacin, sulfazotrim, trimethoprim, streptomycin, cefoxitin, vancomycin and enrofloxacin. Elevated CPS counts were observed with an average of >6 log cfu/g. Of the 355 isolates, 177 (49.86%) were identified as S. aureus. Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus coagulans were identified in 3 (0.84%), 2 (0.56%), 2 (0.56%), and 1 (0.28%) isolates, respectively. Of the total number of S. aureus, 25 (52.08%) were positive for the gene that encodes for toxic shock toxin (TSST-1). Another 16 (33.33%) were positive for the sea gene, and 4 isolates (8.33%) were positive for see and one isolate each was positive for seb (2.08%), sec (2.08%), and etb (2.08%) genes. All isolates demonstrated lipolytic activity under mesophilic and psychrotrophic conditions. S. intermedius and S. hyicus had the most prominent proteolytic potential. Multidrug resistance was observed in most of the potentially toxigenic isolates, with clindamycin having the lowest efficiency (40%), whereas the aminoglycosides (gentamicin and streptomycin) had the highest effectiveness demonstrating inhibition in all evaluated isolates. Methicillin-resistant S. aureus (MRSA) was detected. Minas Frescal cheeses, marketed in the north of Tocantins in the Brazilian Amazon region, do not comply with legal quality standards and pose a public health risk due to the enterotoxigenic potential of multiresistant isolates, in addition to low shelf life of the samples given the high spoilage potential of this microbiota.

A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA

Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2–3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50–60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.

Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

Role of Circulating Tumor DNA As a Prognostic Biomarker in Patients with Diffuse Large B-Cell Lymphoma

Introduction: Nearly 50-60% of the patients withDiffuse large B-cell lymphoma (DLBCL) can be cured by front line chemoimmunotherapy. Current risk stratification modalities include cell-of-origin (germinal center (GCB) or non-GCB (activated B-cell [ABC])) subtyping, use of the revised International Prognostic Index (R-IPI), Ki-67 proliferation index assessment, and cytogenetic aberration detection. However, a novel approach for molecular residual disease (MRD) detection and treatment monitoring is needed to improve patient outcomes. Methods: A personalized and tumor-informed multiplex PCR assay (Signatera™ bespoke mPCR NGS assay) was used for the detection and quantification of ctDNA in a prospective clinical cohort of patients with DLBCL. Serial ctDNA testing was performed at various time points collected at the treating physician's discretion, including pretreatment (baseline), during first-line therapy, during and post-salvage therapy, at the end of treatment, and during surveillance, up to the last clinical follow-up. ctDNA clearance was defined as change in ctDNA status (positive to negative) from the pre-treatment time point to the subsequent time point. Event-free survival (EFS) was defined as the time elapsed since the initial ctDNA clearance or PET imaging, for those without ctDNA clearance, during which the patient did not experience progression, death or treatment with salvage therapy. EFS was censored at the last date of followup if the patient was alive and without an event. Overall survival (OS) was defined as the elapsed time after primary treatment until death from any cause. Univariate and multivariable models were analyzed using Cox proportional models. Results: In this retrospective evaluation of commercial ctDNA testing in patients with stage I-IV DLBCL, a total of 50 patients (I:3, II:12, III: 4, and IV: 29;median age: 59 years) were analyzed. Patients were followed up for a median of 12.68 months (inter quartile range: 7.9-17.7 months). Of the 50 patients, 41 had pre-treatment (baseline) time points available and ctDNA was detected in 95% (39/41) of patients. Since the majority of the patients had stage IV disease, median ctDNA detection levels were observed to be high (overall median: 534 MTM/ml, range: 0-5957 MTM/ml; I median: 9.6 MTM/mL [range: 0.2-2822 MTM/mL], II median: 53 MTM/mL [range: 0.8-862 MTM/mL], III median: 368 [range: 2-940 MTM/mL], IV median: 1011 MTM/mL [range: 74-5958 MTM/mL]). Clearance of ctDNA at any point during first-line therapy was associated with improved outcomes (EFS, HR: 8.3, 95% CI: 2.56-27.0, p<0.0001 and OS, HR: 15.2, 95% CI: 1.53-149, p=0.0021), compared to patients who remained ctDNA-positive. Furthermore, 48% (13/27) of patients were found to clear their ctDNA upon the completion of the first cycle of treatment. On comparing baseline ctDNA levels with other clinicopathological risk factors, high ctDNA levels were observed to be strongly correlated with higher R-IPI scores (0-2 vs. 3-5; p=0.0008) and higher stage (I-II vs. III-IV; p=0.0007). Additionally, multivariate analysis determined that ctDNA clearance significantly outperformed all other clinicopathologic risk factors associated with EFS (HR: 48.8, 95% CI:1.2-2005, p=0.04). Finally, ctDNA clearance predicted complete response (CR)/no evidence of disease (NED) on average 95 days (range: 0-14.7 months) ahead of PET imaging or biopsy. Conclusions: In this study utilizing a real-world cohort of patients with stage I-IV DLBCL, ctDNA clearance during first-line therapy was prognostic for EFS and OS and preceded clinical response. ctDNA testing in patients with DLBCL may enable personalized surveillance, intervention, and/or trial options, ultimately improving patient outcomes.

Detection of virulence genes and multidrug resistance in Pseudomonas aeruginosa clinical isolates from a public hospital in Lagos, Nigeria

Pseudomonas aeruginosa is a main cause of nosocomial infections and is classified as a priority critical pathogen of global concern. Multidrug resistance and virulence genes attributed to the pathogenicity of the organism has resulted in failed therapies, linked to increased morbidity and mortality in infected patients. The aim of this study was to determine the virulence genes and multidrug resistant P.aeruginosa in clinical specimens from a secondary health care center laboratory in Lagos, Nigeria. MethodologyA total of three hundred and sixty-seven clinical samples were examined with conventional methods and Microbact GNB12A. Forty- seven (47) P. aeruginosa strains were identified. Antimicrobial susceptibility test was performed by the standard disk diffusion method following CLSI guidelines. Identification of P. aeruginosa was done by amplification of oprL and oprI genes. Monoplex and multiplex PCR assays were used for detecting virulence genes (lasB, toxA, exoU and nan1). Descriptive statistical methods were used to analyze the distribution of P. aeruginosa by sample sources, MDR patterns by number of isolates and virulence gene carriage by MDR (≥3classes of antibiotics). ResultsA total of forty-seven (47) isolates were confirmed as P. aeruginosa. The isolates showed pan-resistance to cefuroxime and ceftazidime. An elevated level of resistance was also seen in amoxycillin-clavulanic acid, ceftriaxone, cefixime and meropenem. Susceptibility to Amikacin was 95.7 %. Multidrug resistance was seen in forty (85.1 %) of the isolates. P. aeruginosa (100 %) were confirmed with oprL and 74.5 % oprI genes. All isolates (100 %) had lasB, toxA and exoS virulence genes while nan1 was detected in 19.1 % isolates. ConclusionsDiverse and multiple virulence genes were distributed among the multidrug resistant isolates. Understanding the factors contributing to pathogenicity and antibiotic resistance will aid in the treatment of infection. It will also promote judicious use of antimicrobials.

586P Clinical validation of a multiplex polymerase chain reaction (mPCR) assay to identify patients (pts) with NSCLC suitable for mobocertinib treatment

586P Clinical validation of a multiplex polymerase chain reaction (mPCR) assay to identify patients (pts) with NSCLC suitable for mobocertinib treatment

Unveiling Hidden Risks: Intentional Molecular Screening for Sexually Transmitted Infections and Vaginosis Pathogens in Patients Who Have Been Exclusively Tested for Human Papillomavirus Genotyping.

This analytical, prospective, cross-sectional study included 408 males and females. Eligible participants had positive and negative HPV genotyping test results and agreed to early detection or had HPV antecedents. They provided the same urogenital samples used for HPV detection and, through our multiplex in-house PCR assay, we screened for Candida spp., Ureaplasma spp., Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, herpes simplex virus 1 and 2 (HSV), Mycoplasma spp., molluscum contagiosum virus (MCV), Treponema pallidum, Haemophilus spp., Staphylococcus aureus, and Klebsiella spp. The subsequent statistical analysis aimed to reveal correlations between HPV genotypes and the identified pathogens. Of the participants, 72.1% (n = 294) tested positive for HPV genotypes. HR-HPV (high-risk HPV) genotypes comprised 51 (8.1%), 66 (7.1%), and 58 (6.1%). Haemophilus spp., Ureaplasma spp., Candida spp., Staphylococcus aureus, and Mycoplasma spp. frequently co-occurred with HPV infection (p < 0.05). Gender-based variations were notorious for Ureaplasma spp., Mycoplasma spp., and MCV (p < 0.05). Coinfections were prevalent (43.9%), with a positive HPV result elevating the risk for Trichomonas vaginalis, Mycoplasma spp., Staphylococcus aureus, HSV, and MCV (OR > 1, p < 0.05). HPV 16 correlated with HSV and Ureaplasma spp., while HPV 6 was linked with HSV and MCV (p < 0.05). This screening strategy uncovered significant coinfections and associations between HPV genotypes and pathogens, underscoring the importance of routine screening to explore clinical implications in urogenital health.

Nasal MRSA carriage is a risk factor for development of antibiotic resistance in diabetic foot ulcers and is significantly higher than diabetic and non-diabetic individuals without foot ulcer

BackgroundDiabetic foot ulcer (DFU) is a major complication of diabetes often impacted by polymicrobial infection in the wound site. Diabetic patients are immunocompromised in nature and hence vulnerable to infection once the skin barrier is breached. Microbiological culture-based methods show that Staphylococcus aureus (SA) is the most frequently isolated bacteria from the DFU wounds. SA and its most clinically important antibiotic resistant variant methicillin-resistant S. aureus (MRSA) are commonly found in the nasal vestibule and colonization of SA as well as MRSA in any wound site can aggravate the condition. We hypothesize that the presence of nasal MRSA carriage can serve as a potential risk factor contributing to the emergence of antibiotic resistance in diabetic foot ulcer wounds.MethodsIn the present study, we have compared the carriage of SA and MRSA in nasal cavity and foot skin among DFU patients (D+F+, n = 50), diabetic patients without any ulcer (D+F-, n = 50), and healthy controls (D-F-, n = 40) by using bacterial culture and PCR based methods. The D+F+, D+F- and D-F-individuals were further categorized based on the presence or absence of MRSA and clinical parameters were compared between MRSA+ ve and MRSA-ve individuals in each of the three groups mentioned above.ResultsOur results show that, (a) nasal MRSA carriage is significantly higher (p < 0.05) in D+F+ group than the D+F- and D-F- and significantly associated with wound MRSA carriage in D+ F+ individuals (O.R. = 4.09; 95% C.I. = 1.12–15.05) and (b) the HbA1C level is significantly higher (p < 0.02) in wound MRSA positive, compared to MRSA negative D+F+ patients. Interestingly more than half of the MRSA (64%) isolated from DFU wound were identified to be multidrug resistant.ConclusionThese findings strongly suggest that nasal MRSA carriage can act as a risk factor for development of antibiotic resistance in diabetic foot ulcers and it is therefore important to screen nasal and wound sites of these patients regularly. We have also developed a rapid multiplex PCR assay to detect MRSA from clinical isolates or microbial DNA isolated from clinical samples in the hospital settings.

Multiplex high resolution melt curve real-time PCR for detection of Shiga-toxin producing and blaCTX-M-harboring E. coli in beef products

This study aimed to develop a rapid multiplex high resolution melt-curve (HRM) qPCR with an internal amplification control (IAC) for the simultaneous detection of Shiga-toxin producing Escherichia coli (STEC) and blaCTX-M-carrying E. coli in beef. Specific primer pairs targeting eaeA, uidA, stx1, stx2 and blaCTX-M genes were designed. The specificity of the optimized assay was validated using genomic DNA from Gram-positive, Gram-negative, and STEC (non-O157 and O157:H7) strains. The accuracy of the assay for detecting blaCTX-M was confirmed using genomic DNA from bacteria previously reported to carry this gene. Beef products were inoculated with a cocktail of E. coli O157:H7 and E. coli M-1, an isolate reported to harbor blaCTX-M, at concentrations of 10, 100, and 1000 CFU/mL. Among non-STEC DNA samples (n = 45) tested, no target genes were detected in 32 isolates. However, the assay was able to detect uidA and blaCTX-M in seven and six bacterial isolates, respectively. Among the STEC bacterial isolates (n = 57), at least two or more target genes were detected in 50 STEC isolates. All isolates carried the eaeA gene, 55 had stx1, 26 had stx2, and 26 had both stx genes. The uidA gene was detected in all non-O157:H7 isolates, whereas no E. coli O157:H7 isolates was found to be positive for uidA. The blaCTX-M gene was not detected in any of the STEC isolates tested but it was detected in 20 of 23 DNA samples from isolates previously verified as positive for the gene, thus demonstrating the technique's accuracy. Enrichment times ranging from 4 to 8 h were required to achieve a detection limit of 10 CFU/mL in 325 g of inoculated beef. The multiplex HRM real-time PCR assay can be used as a rapid, yet specific, tool for routine examination of STEC and blaCTX-M-harboring bacteria that are present in low concentrations in beef products.

First identification of the SARS-COV-2/XBB.1.5 sublineage among indigenous COVID-19 cases through the influenza sentinel surveillance system in Niger

The emergence of the Omicron variant in November 2021, has caused panic worldwide due to the rapid evolution and the ability of the virus to escape the immune system. Since, several Omicron sublineages (BA.1 to BA.5) and their descendent recombinant lineages have been circulating worldwide. Furthermore, in December 2022, a new Omicron subvariant XBB.1.5 characterized by an unusual mutation in the spike protein evolved in the United States and rapidly spread to the other continents. Our study reports on the first cases of XBB.1.5 sublineage among indigenous Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2) positive cases detected through the influenza sentinel surveillance system in Niger. All influenza suspected cases were tested for both influenza and SARS-COV-2 using the Centre for Disease Control and prevention (CDC) Influenza SARS-COV-2 Multiplex quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) Assay. SARS-COV-2 positive samples with cycle threshold ≤28 were selected for whole genome sequencing subsequently using the Oxford Nanopore Midnight protocol with rapid barcoding on a MinIon MK1B device. A total of 51 SARS-COV-2 positive samples were confirmed between December 2022 and March 2023. We successfully obtained 19 sequences with a predominance of the XBB.1/XBB.1.5 sublineages (73.7 %). In addition, a recombinant XBD sequence was also first-ever identified in early March 2023. Our findings support the need to strengthen the influenza sentinel surveillance for routine Coronavirus Disease 2019 (COVID-19) surveillance and SARS-COV-2 variants monitoring in Niger.

Establishment of a multiplex polymerase chain reaction assay for the detection of marine harmful algae

Establishment of a multiplex polymerase chain reaction assay for the detection of marine harmful algae

Molecular detection of genes encoding resistance to tetracycline and quinolones among Shigella strains isolated from children with acute diarrhea in southwest Iran.

An increase in the antibiotic resistance of Shigella isolates has caused major global challenges in antimicrobial therapy. Knowledge of local antibiotic resistance trends is essential for selecting appropriate antibiotic treatment regimens. This study aimed to evaluate the frequency of efflux-mediated tetracycline resistance (tet) and plasmid-mediated quinolone resistance (qnr) genes among Shigella isolates. This survey investigated 91 Shigella isolates, obtained from children with acute diarrhea. The isolates were identified using standard biochemical tests and confirmed by polymerase chain reaction (PCR) assay. Besides, the susceptibility of isolates to six selected antibiotics was assessed by the disk diffusion method. All tetracycline-resistant and nalidixic acid and ciprofloxacin resistant strains were screened for tet and qnr genes by a multiplex PCR assay. According to the results of antibiotic susceptibility tests, the highest level of antibiotic resistance was related to tetracycline (80.2%) and doxycycline (78.1%), respectively. All isolates were sensitive to tigecycline. The PCR results showed that 40.6%, 3.1%, 21.8%, 61.6% and 28.7% of the isolates carried qnrA, qnrB, qnrS, tetA, and tetB genes, respectively. None of the isolates contained tetC and tetD genes. The current findings revealed that tetA and qnrA genes might play a key role in conferring tetracycline and quinolone resistance.

Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-de- tecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.&#x0D; Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla , AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic iden- tification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilu- tions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.&#x0D; Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL pro- ducers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla , (85%) aac (6́)-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.&#x0D; Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detec- tion rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. bauman- nii in these clinical isolates showed frequent commonality of genotypes.

MecA and fdh: markers of pathogenicity and commensalism in Staphylococcus epidermidis of pediatric origin from Pakistan

Staphylococcus epidermidis is an opportunistic bacterial pathogen. The study screened isolates of S. epidermidis of pediatric origin for genetic markers of discriminatory potential. 103 isolates (n = 75 clinical; n = 28 community) were screened for methicillin resistance (mecA), formate dehydrogenase (fdh) and an array of virulence factors through multiplex PCR and Congo red assay. The isolates were typed in four distinct categories, based on the presence of selected virulent factors. The type A clinical isolates carrying icaADBC operon (n = 22; 29.3%, P = 0.117) were not significantly differentiating the origin of isolates. The type B clinical isolates representing methicillin resistant S. epidermidis (MRSE) (n = 73; 97.3%, P < 0.00001) and the type C clinical isolates lacking formate dehydrogenase fdh (n = 62; 82.6%, P < 0.00001) were having significant discriminatory potential of clinical isolates, respectively. All type D community isolates were carrying fdh (n = 28; 100%, P < 0.00001). MecA and fdh are significant differential markers of pathogenicity and commensalism in S. epidermidis of pediatric origin.

Development of a Multiplex PCR Assay for Efficient Detection of Two Potential Probiotic Strains Using Whole Genome-Based Primers.

Probiotics are microorganisms that exert strain-specific health-promoting effects on the host. Τhey are employed in the production of functional dairy or non-dairy food products; still, their detection in these complex matrices is a challenging task. Several culture-dependent and culture-independent methods have been developed in this direction; however, they present low discrimination at the strain level. Here, we developed a multiplex PCR assay for the detection of two potential probiotic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum L125 and Lp. pentosus L33, in monocultures and yogurt samples. Unique genomic regions were identified via comparative genomic analysis and were used to produce strain-specific primers. Then, primer sets were selected that produced distinct electrophoretic DNA banding patterns in multiplex PCR for each target strain. This method was further implemented for the detection of the two strains in yogurt samples, highlighting its biotechnological applicability. Moreover, it can be applied with appropriate modifications to detect any bacterial strain with available WGS.

A Novel Nested Multiplex Polymerase Chain Reaction Assay for Malaria Diagnosis Using the Hydroxymethyl Dihydropterin Pyrophosphokinase-Dihydropteroate Synthase (hppk-dhps) Gene.

There are many techniques for malaria diagnosis. Currently, the nested polymerase chain reaction (PCR) method based on a small subunit ribosomal RNA gene (18S rRNA) has been used as a confirmatory method. However, this method is time-consuming, laborious, and costly. Therefore, the objective of this study was to develop nested multiplex PCR for Plasmodium species identification using the dihydropterin pyrophosphokinase-dihydropteroate synthase (hppk-dhps) gene. Genus- and species-specific primers for the hppk-dhps gene were designed. The performance of the novel nested multiplex PCR was compared with 18S rRNA nested PCR. A total of 115 blood samples were used in this study, including 84 infected samples and 31 uninfected samples. Analysis of the blood samples by nested multiplex PCR targeting the hppk-dhps gene identified 81 infected cases. The level of agreement between this novel method and 18S rRNA nested PCR was 97.4%. Further, the novel method successfully detected all human malaria parasites except Plasmodium ovale and detected mixed Plasmodium falciparum/Plasmodium vivax infections. The sensitivity and specificity obtained from this novel method were 96.4% and 100%, respectively. The limit of detection of the hppk-dhps nested multiplex PCR for P. falciparum and P. vivax was 500 parasites/µL and 4 parasites/µL, respectively. The lowest parasite gDNA detected by this method was 0.5 ng/µL for P. falciparum and 0.1 ng/µL for P. vivax. These results corroborate that the hppk-dhps gene is a novel amplification target for the detection of human malaria. This novel target PCR-based method is a beneficial approach for malaria diagnosis, as well as species identification and differentiation.

A rapid, multiplex digital PCR assay to detect gene variants and fusions in non-small cell lung cancer.

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1 and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target-signal encoding to improve the specificity of variant detection by reducing background noise by up to an order of magnitude. The assay reported an overall 100% positive percent agreement (PPA) and 98.5% negative percent agreement (NPA) compared to a sequencing-based assay in a cohort of 62 human formalin-fixed paraffin-embedded (FFPE) samples. In addition, the dPCR assay rescued actionable information in ten samples that failed to sequence, highlighting the utility of a multiplexed digital PCR assay as a potential reflex solution for challenging NSCLC samples.

A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

Prevalence of Foodborne Bacterial Pathogens and Antibiotic Resistance Genes in Sweets from Local Markets in Iran.

This study aimed to investigate the prevalences of some important antibiotic-resistance genes (ARGs) and foodborne bacterial pathogens in sweet samples collected from local markets in Iran. Forty sweet samples were collected. Foodborne pathogens and ARGs were detected in the sweet samples by conventional and multiplex PCR assays using species-specific primers. Staphylococcus aureus, Cronobacter sakazakii, Shigella spp., Campylobacter jejuni, and Campylobacter coli were detected and identified in 47.5%, 20%, 45%, 5%, and 30% of the sweet samples, respectively. We found S. aureus and Shigella spp. were the most prevalent bacterial pathogens. S. aureus was found to be the most frequent pathogenic bacteria profiled in these samples. We also found a significant correlation between the presence of C. coli and Cr. sakazakii. We detected the blaSHV resistance gene in 97.5% of the sweet samples; however, blaTEM was detected in only one sample (2.5%). Regarding these results, we suggest preventive strategies such as implementing automation of food processing; monitoring the personal hygiene and health of food handlers, and testing regularly for antibiotic resistance in raw materials and products.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.