Real-time PCR Kit Research Articles

Evaluation of the Vaginal Panel Realtime PCR kit (Vircell, SL) for diagnosing vaginitis: A comparative study with routinely used diagnostics.

Vaginitis is a prevalent clinical disorder associated with several adverse health consequences, prompting women to seek medical care. In this study we evaluate the Vaginal Panel Real-Time PCR kit (qPCR test) against routinely used diagnostics for detection of bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and trichomoniasis. A total of 1011 vaginal swab specimens were analyzed. The routinely diagnostic methods for BV was Gram stain-based Nugent score. VVC presence was detected by culture, and Candida species were identified using MALDI-TOF MS. Trichomonas vaginalis was identified by culture in a selective medium. Molecular analyses were conducted on the MagXtract® 3200 System and analyzed using the CFX96™ Real-Time PCR Detection System. The sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR test compared to the reference method for BV diagnosis was 93.1%, 88.8%, 90.1% and 92.2%, respectively, with a Kappa value of 0.82. For Candida species, sensitivity, specificity, positive predictive value, and negative predictive value were 96.0%, 98.4%, 95.3%, and 98.7%, respectively. The qPCR test detected 32 additional positive samples for Candida not reported by the routinely used diagnostics. For trichomoniasis, the qPCR test identified T. vaginalis in fifteen specimens, despite no microscopic detection in cultured specimens. Our results demonstrate that the Vaginal Panel Real-Time PCR kit shows optimal concordance with routinely used diagnostics for diagnosing vaginitis. Furthermore, enhancing detection of T. vaginalis. However, further validation studies are necessary to confirm its full diagnostic accuracy. The use of nucleic acid amplification tests (NAATs) provides rapid and accurate diagnosis, crucial for early detection and treatment of vaginitis.

Development of a new tool to detect Melampsora medusae f.sp. tremuloidae causing rust disease on Populus tremuloides.

Melampsora medusae f. sp. tremuloidae is a quarantine organism for the EU. In North America, this fungus causes rust disease on Populus tremuloides. In Europe, Populus tremula, an aspen closely related to P. tremuloides, is widespread and plays an important ecological role. Introduction of M. medusae f. sp. tremuloidae into Europe could be a major risk if this forma specialis could evolve and become virulent on P. tremula. To date no PCR-based assay exists to specifically detect M. medusae f. sp. tremuloidae. In this study, a sensitive and specific real-time PCR assay has been developed based on the 28S rDNA. The assay proved to be reliable using many real-time PCR kits and platforms. It can be used to monitor the introduction and the spread of M. medusae f. sp. tremuloidae in the context of phytosanitary regulations.

Molecular characterization of human adenovirus associated with pediatric severe acute respiratory infections in a tertiary care hospital in North East India

PurposeThe present study explored the molecular characterization of human Adenovirus (HAdV) and its strains among hospitalized SARI cases in the pediatric unit of a tertiary care hospital in North-East India.MethodsNasal and throat swabs were collected from 70 patients of Pediatric Unit, of a tertiary hospital in NE India from April 2023-October 2023. The samples were screened for the presence of HAdV using an adenovirus-specific Real-Time PCR Kit. For molecular characterization, Next Generation Sequencing (NGS) was performed by targeting the hexon gene of HAdV followed by post-sequencing analysis.ResultsOverall, 18.57% (13/70) of samples were positive for HAdV. In context of the severity of illness, 3/13 adenovirus-positive patients (23.07%) died after hospitalization, had severe pneumonia among which two were of less than one year of age. Molecular characterization using NGS indicated that 4/13 individuals were infected with HAdV-B type 3 and 5/13 patients were infected with HAdV type 7. Notably, 4/7 cases of severe pneumonia were under five years of age and associated with HAdV type 7 infection. The ratio of non-synonymous to synonymous mutation (dN/dS) was comparatively low in HAdV type 7 positive samples (dN/dS=0.31). No non-synonymous mutation was observed in HAdV-B type 3 positive samples. The higher neutrophil percentage among the death cases suggested an acute immune response.ConclusionThe study demonstrated HAdV type 7 and HAdV-B type 3 as strains associated with pediatric SARI cases from April 2023-October 2023. Further, HAdV type 7 infection was primarily linked with lower respiratory tract infections mainly severe pneumonia.

Assessment of Nine Real-Time PCR Kits for African Swine Fever Virus Approved in Republic of Korea.

The African swine fever virus (ASFV) causes severe disease in wild and domestic pigs, with high mortality rates, extensive spread, and significant economic losses globally. Despite ongoing efforts, an effective vaccine remains elusive. Therefore, effective diagnostic methods are needed to rapidly detect and prevent the further spread of ASF. This study assessed nine commercial kits based on real-time polymerase chain reaction (PCR) approved in the Republic of Korea using the synthesized ASFV plasmid, 20 food waste samples, and artificially spiked samples (ASSs). The kits were evaluated for their diagnostic sensitivity, specificity, cost per reaction, and reaction running time. In addition, the results were compared with those of the World Organization for Animal Health (WOAH) standard methods. Three commercial kits (VDx® ASFV qPCR Kit, Palm PCR™ ASFV Fast PCR Kit, and PowerChek™ ASFV Real-time PCR Detection Kit Ver.1.0) demonstrated the highest sensitivity (100 ag/μL), cost-effectiveness (less than KRW 10,000), and shortest running time (less than 70 min). These kits are suitable for the monitoring, early diagnosis, and prevention of the spread of ASF. This is the first report on the performance comparison of ASFV diagnostic kits approved in the Republic of Korea, providing valuable information for selecting kits for testing with food waste samples.

Comparison of qPCR and chromogenic culture methods for rapid detection of group B streptococcus colonization in Vietnamese pregnant women

IntroductionNeonatal infections can rapidly become severe, with delays in treatment often proving fatal. Streptococcus agalactiae (Group B Streptococcus, GBS) is a common cause, typically transmitted from colonized pregnant women to neonates during childbirth. In Vietnam, routine prenatal care lacks standardized GBS screening protocols. This study aims to compare enhanced qPCR methods with the culture method, evaluate the diagnostic accuracy of these qPCR procedures, and assess the frequency of GBS infection in pregnant Vietnamese women during their final trimester. Materials and methodsPregnant women aged 35 weeks gestation or more were recruited. Rectovaginal swabs were collected and analyzed for GBS using chromogenic culture, a commercial real-time PCR kit, and in-house real-time PCR assays targeting the cfb and sip genes. Clinical diagnostic values were calculated, and GBS prevalence was determined. ResultsThe study included 259 pregnant women with a mean age of 30.2 ± 5.0 years. Of these, 96.6 % had gestational ages of 37 weeks or more at delivery. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the cfb-based and sip-based qPCR assays were 94.1/92.7, 99.0/99.5, 97.1/98.5, 97.8/97.3, and 97.6 %, respectively. The Kappa values were excellent (0.940 and 0.939), with results available in under 2 h. GBS prevalence was 24.7 % and 25.5 % by cfb-based and sip-based qPCR assays, aligning with the culture method (25.5 %). ConclusionsBoth direct real-time PCR assays demonstrated high accuracy and were comparable to chromogenic culture in diagnosing GBS. A significant prevalence of GBS colonization was found among Vietnamese pregnant women in their final trimester.

Diagnostic Accuracy of Five Molecular Assays for the Detection of Dengue Virus.

Background and Objectives: The steady spread of dengue virus (DENV) poses a profound public health threat worldwide. Reverse transcription real-time polymerase chain reaction (RT2-PCR) has been increasingly recognized as a reference method for the diagnosis of acute dengue infection. The goal of this study was to assess the diagnostic accuracy of five different RT2-PCR kits for the detection of DENV in a historically processed set of sera samples. Materials and Methods: In this retrospective study, 25 sera samples from routinely processed unique adult patients with a known DENV status (previously tested in both molecular and serological assays) were tested in parallel using four conventional (RealStar Dengue PCR Kit 3.0, Clonit'ngo Zika, Dengue & Chikungunya, BioPerfectus Zika Virus/Dengue Virus/Chikungunya Virus Real Time PCR Kit and Novaplex Tropical fever virus) and one sample-to-result (STANDARD M10 Arbovirus Panel) RT2-PCR assays. Additionally, an end-point dilution analysis was conducted in quintuplicate on six serial dilutions of an RNA preparation obtained from a culture-grown DENV serotype 1 strain for a total of 150 tests. Results: The overall accuracy of the evaluated tests ranged from 84% to 100%. In particular, the sensitivity of three conventional RT2-PCR assays (RealStar, Clonit'ngo and Novaplex) was 100% (95% CI: 79.6-100%), while it was lower (73.3%; 95% CI: 48.1-89.1%) for the BioPerfectus kit. The sample-to-result STANDARD M10 panel performed comparatively well, showing a sensitivity of 92.9% (95% CI: 68.5-98.7%). No false positive results were registered in any assay. The end-point dilution analysis suggested that the RealStar kit had the lowest limit of detection. Conclusions: Available RT2-PCR kits for the detection of DENV are highly specific and generally sensitive and, therefore, their implementation in diagnostic pathways is advisable.

The simultaneous presence of active BK, Epstein Barr, and human cytomegalovirus infection and their correlation by host factors in patients suspected of kidney transplant rejection

AimsThis study aims to evaluate the presence of EBV, HCMV, and BKV genomic sequences in the plasma samples (active infection/viremia) of kidney transplant recipients suspected of rejection and to investigate host and risk factors related to the activation of these viruses in these patients.MethodsIn this cross-sectional single-center study, plasma samples were collected from 98 suspected kidney transplant rejection patients at Labafinejad Hospital, Tehran, Iran, between December 2022 and June 2023. Quantitative real-time PCR assays for HCMV, EBV, and BK were performed using GeneProof Real-time PCR kits. ROC curve analysis was used to determine the viral load cutoff point for each virus.FindingsHCMV active viremia was detected in 18 (18.36%) recipients, EBV active viremia in 7 (7.14%), and BKV active viremia in 5 (5.10%). ROC results indicated viral load cutoff points of 778, 661, and 457 points for HCMV, EBV, and BKV, respectively. The duration of time after transplantation significantly differed between active viremia and no viremia groups (120.5 vs. 46 months, P = 0.014). In the BKV active viremia group, the increase in creatinine compared to baseline creatinine was significantly higher than in the no viremia group (2.7 vs. 0.8, P = 0.017). The odds ratio of HCMV active viremia in patients taking tacrolimus was 2.84 times higher, and the odds of HCMV active viremia in patients taking antithymocyte globulin was 3.01 times higher than in patients not taking these drugs.ConclusionRapid and timely diagnosis of viral active infections in kidney transplant patients is crucial for effective disease management and implementation of appropriate treatment strategies. Identifying potential risk factors, including host and treatment-related factors that influence transplantation, can facilitate the development of suitable preventive strategies.

Human Papillomavirus Genotypes Distribution in High-Grade Cervical Lesions and Invasive Cervical Carcinoma in Women Living in Mauritania: Implications for Cervical Cancer Prevention and HPV Prophylactic Vaccination.

Cervical cancer related to high-risk human papillomavirus (HR-HPV) is the second female cancer in Mauritania (Northwest Sahelian Africa). We assessed the distribution of HPV genotypes in Mauritanian women with high-grade cervical intraepithelial neoplasia (CIN2/3) or invasive cervical cancer (ICC). A prospective study was conducted in the Centre Hospitalier National, Nouakchott, Mauritania, to collect cervical biopsies among women suspected of CIN2/3 or cancer. HPV DNA detection and genotyping were carried out from formalin-fixed, paraffin-embedded biopsies using multiplex PCR (Human Papillomavirus Genotyping Real-Time PCR Kit, Bioperfectus Technologies Co., Taizhou, China). Fifty biopsies were included from women (mean age: 56.7 years) suffering from CIN2/3 (28.0%) and ICC (72.0%) which corresponded to 32 (64.0%) squamous cell carcinomas (SCC) and 4 (8.0%) adenocarcinomas (ADC). HPV DNA detection was successful in 47 (94.0%) samples. The most prevalent HR-HPV genotypes were HPV-45 (40.4%), HPV-16 (38.3%), HPV-39 and HPV-52 (23.4%), HPV-33 (17.0%), HPV-18 (14.9%), HPV-35 (4.2%), and HPV-56 (2.1%). The majority (93.6%) of HPV-positive biopsies contained at least one HPV type covered by the 9-valent Gardasil-9® vaccine, and 40.9% were infected by multiple vaccine HPV genotypes. To eradicate cervical cancer in Mauritania, prophylactic HPV vaccination must be combined with primary molecular screening of cervical HR-HPV infection.

Epidemiology of childhood enterovirus infections in Hangzhou, China, 2019–2023

Human enteroviruses are highly prevalent world-wide. Up to more than 100 subtypes of enteroviruses can cause several diseases, including encephalitis, meningitis, myocarditis, hand-foot-mouth disease, conjunctivitis, respiratory diseases, and gastrointestinal diseases, thus posing a great threat to human health. This study aimed to investigate the epidemiological characteristics of enterovirus in children in Hangzhou, China before and after the COVID-19 outbreak. Systematic monitoring of enterovirus infections was performed by collecting samples from the children admitted to the inpatient wards and outpatient departments in the Children’s Hospital, Zhejiang University School of Medicine, between January 2019 and May 2023. A commercial real-time RT PCR kit was utilized to detect enteroviruses. Among the 34,152 samples collected, 1162 samples, accounting for 3.4% of the samples, were tested positive for enteroviruses. The annual positive rates of the enteroviruses were 5.46%, 1.15%, 4.43%, 1.62%, and 1.96% in 2019, 2020, 2021, 2022, and May 2023, respectively. The positivity rate of the enteroviruses was highest among children aged 3–5 years and 5–7 years. Moreover, the monthly positivity rate of enterovirus infection ranged from 0.32% to 10.38%, with a peak in June and July. Serotypes, especially EV71 and CA16, causing severe symptoms such as HFMD, were decreasing, while the proportion of unidentified serotypes was on the rise. The incidence of enteroviruses in Hangzhou was higher in children aged 1–3 years and 7–18 years.

Evaluation of Screening Results for Vancomycin-Resistant Enterococci: Three-Year Surveillance.

Enterococci are facultative anaerobic, binary, or chained Gram-positive cocci. The gastrointestinal colonization of hospitalized patients is the most important reservoir of vancomycin-resistant enterococci. We aimed to evaluate retrospectively the screening results of vancomycin-resistant enterococci, studied by the simultaneous (real-time) polymerase chain reaction method on rectal swabs of adult and pediatric patients hospitalized in our hospital in 2019-2021. Adult and pediatric patients were included in our study between Jan 2019 and Dec 2021. The results of vancomycin-resistant enterococci, studied with the real-time polymerase chain reaction method from rectal swabs sent from intensive care units and services, were analyzed retrospectively. Isolation of the samples was performed using the Fluorion VRE QLP 1.0 real-time polymerase chain reaction kit (Iontek, Turkey), and detection was performed with the Fluorion Detection System (Iontek, Turkey) real-time polymerase chain reaction device. Overall, 31,725 patients were included in our study. When evaluated in order of years, in 2019, 379 (7%) of 5,389 adults, 322 (7.4%) of 4,003 children, 234 (5.5%) of 4,185 adults in 2020, 157 (2.4%) of 6,499 children, and in 2021, vancomycin-resistant enterococci were detected in 469 (7.5%) of 6,232 adults and 224 (4.1%) of 5,417 children. The prevalence of vancomycin-resistant enterococci is greater in adults, particularly in intensive care units, compared to children. Infection control precautions and training be augmented in high-risk clinics, while the unnecessary utilization of glycopeptides should be limited.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis.

PEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis, are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens. Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of B. hyodysenteriae, and the 16S rDNA gene of L. intracellularis, specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for B. hyodysenteriae and L. intracellularis, and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, B. hyodysenteriae and L. intracellularis. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae, and 98.6% for L. intracellularis. The co-infection rates were 16.85% for PEDV + B. hyodysenteriae, 31.46% for PEDV + L. intracellularis, 57.86% for B. hyodysenteriae + L. intracellularis, and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis, respectively. The new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.

Agreement between two real-time commercial PCR kits and an in-house real-time PCR for diagnosis of mucormycosis.

Early diagnosis of mucormycosis is crucial for initiating effective treatment. The detection of Mucorales DNA by PCR in serum has revolutionized the diagnosis of this infection. However, the use of in-house methods can be time consuming. The availability of a commercial kit eliminates the need for in-house assay development, reducing laboratory workload and ensuring consistent performance across different healthcare settings. Currently, there are several commercial assays available, but many have limited evaluation. In this study, we compared two commercial kits and found that the MycoGENIE Kit offers a promising alternative to the in-house method.

MOLECULAR IDENTIFICATION OF HCV GENOTYPES AMONG INJECTING DRUG USERS HAVING HCV and HIV CO-INFECTION

Co-infection with hepatitis C virus (HCV) and Human immunodeficiency virus (HIV) is common in Injecting drug users (IDUs). The aim of this study was the molecular identification of HCV genotypes in IDUs having HC/HIV co-infection in Peshawar. A cohort cross-sectional study was conducted in Nai Zindagi NGO from 2020 to 2022. A sample of 350 IDUs including 309 males, 23 females, 09 children, and 09 transgender were enrolled. Suspected age was 34 years. Screening of HIV and HCV infection was performed through ICT and RT-PCR. For genotype determination, a specific SACACE real-time PCR kit was used. Out of a total of 350 patients, 204 were HCV/HIV co-infected. According to bivariate analysis, there is statistically moderate positive r=522 between viral load and HCV/HIV co-infection (p=0.000). It is concluded that the prevalence of HCV/HIV co-infection was 44.28% in IDUs with the prevalent genotype 3a (51.1%). Viral load of males was higher than females. To overcome the burden of HCV/HIV co-infection large-scale, multicentre, and multistate studies should be conducted across Pakistan and preventive measures should be taken to reduce the use of syringes, razors, tattooing, sex workers, and blood transfusion.

Efficacy of a novel, multiplex, real-time PCR kit for the detection of Helicobacter pylori and clarithromycin resistance

Efficacy of a novel, multiplex, real-time PCR kit for the detection of Helicobacter pylori and clarithromycin resistance

Preliminary study on the diversity and quantity analysis of oral bacteria according to the sampling methods

Objectives: Oral bacterial samples included subgingival, supragingival, and saliva plaques. As the diversity and number of microorganisms deffer depending on the area of the oral cavity and the method used, an appropriate and reliable collection method is important. The present study investigated oral bacterial sampling methods. Methods: Supragingival dental plaque was collected from the buccal and lingual tooth surfaces of study participants using sterilized cotton swabs. Plaques were collected from the subgingival area using a sterilized curette. Bacterial genomic DNA was extracted using MagNA Pure 96 DNA and Viral NA low-volume kits. Real-time polymerase chain reaction (PCR) was performed using the PowerCheck™ Periodontitis Pathogens Multiplex Real-time PCR kit. Results: Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum of the orange complex were not observed in the subgingival biofilms of all study participants. For Porphyromonas. gingivalis, a significant correlation was observed between supragingival, subgingival, and total tooth surface biofilms. Compared to the supragingival and subgingival biofilmss, total tooth surface biofilm exhibited the highest bacterial count when the inswabbing method was used. Conclusions: Based on these findings, the supragingival swab method is recommended for oral bacterial research.

Correlation between the Cycle Threshold Values in Detection of Severe Fever with Thrombocytopenia Syndrome Virus Using PowerChekTM SFTSV Real-Time PCR Kit and Viral Load: Prognostic Implications.

This study aimed to analyze the correlation between the cycle threshold (Ct) values of severe fever with thrombocytopenia syndrome (SFTS) virus small (S) and middle (M) segments and the SFTS viral load, aiming to estimate the initial viral load and predict prognosis in the early clinical course. A retrospective study was conducted with confirmed SFTS patients at Jeju National University Hospital (2016-2022). Patients were categorized into non-fatal and fatal groups. This study included 49 patients with confirmed SFTS (non-fatal group, n = 42; fatal group, n = 7). A significant negative correlation (-0.783) was observed between the log SFTS viral load and Ct values (p < 0.001). This negative correlation was notably stronger in the fatal group (correlation coefficient -0.940) than in the non-fatal group (correlation coefficient -0.345). In this study, we established a correlation between SFTS viral load and Ct values for estimating the initial viral load and early predicting prognosis. These results are expected to offer valuable insights for SFTS patient treatment and prognosis prediction.

Epidemiology of Spinal Muscular Atrophy Based on the Results of a Large-Scale Pilot Project on 202,908 Newborns

BackgroundThis study presents the findings of a newborn screening (NBS) pilot project for 5q-spinal muscular atrophy (5q-SMA) in multiple regions across Russia for during the year 2022. The aim was to assess the feasibility and reproducibility of NBS for SMA5q in diverse populations and estimate the real prevalence of 5q-SMA in Russia as well as the distribution of patients with different number of SMN2 copies. MethodsThe pilot project of NBS here was based on data, involving the analysis of 202,908 newborns. SMA screening assay was performed using a commercially available real-time polymerase chain reaction kit, the Eonis SCID-SMA. ResultsIn one year, 202,908 newborns were screened, identifying 26 infants with homozygous deletion of SMN1 exon 7, yielding an estimated 5q-SMA incidence of 1:7804 newborns. It was found that 38.46% had two SMN2 copies, 42.31% had three copies, 15.38% had four copies, and 3.85% had five copies of SMN2. Immediate treatment was proposed for patients with two or three SMN2 copies. Infants with four or more SMN2 copies warranted further investigation on management and treatment. Short-term monitoring after gene therapy showed motor function improvements. Delays in treatment initiation were observed, including the testing for adeno-associated virus 9 antibodies and nonmedical factors. ConclusionsThe study emphasizes the need for a standardized algorithm for early diagnosis and management through NBS to benefit affected families. Overall, the NBS program for 5q-SMA in Russia demonstrated the potential to improve outcomes and transform SMA from a devastating disease to a chronic condition with evolving medical requirements.

Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses.

Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses. A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay. This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/μL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively. The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.

Evaluation of a new CT/NG/TV/MG Real-Time PCR Kit (Vircell) versus the Allplex STI Essential Assay (Seegene) for the diagnosis of sexually transmitted infections.

Sexually transmitted infections (STI) are a public health problem. Real-time PCR assays are the most sensitive test for screening and diagnosis of these infections. The aim of this study was to evaluate a new CT/NG/TV/MG Real-Time PCR (RT-PCR) kit (Vircell) for the detection of Chamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis for the diagnosis of sexual transmitted infections using the Allplex STI Essential Assay (Seegene) as the reference's method. A total of 497 samples from different anatomical sites (endocervical, urethral, rectal, pharyngeal and urine) were analysed from October 2022 to February 2023. A total of 108 (21.73 %) and 106 (21.33 %) positive samples were found for any of the assays used. The most commonly detected pathogen was N. gonorrhoeae (52 samples; 10.46 %), and the least commonly detected was T. vaginalis (three samples; 0.60 %). The anatomical site with the highest prevalence of micro-organisms was a non-urogenital site, the pharynx (26 positive samples; 5.23 %). Using the Allplex STI Essential Assay (Seegene) as the reference method, the diagnosis performance showed that the average specificity of CT/NG/TV/MG RT-PCR Kit (Vircell) was 99.84 % and the sensitivity was 99.53 %. The overall concordance was k=0.98 (CI95 %; 0.96-1). In conclusion, the CT/NG/TV/MG RT-PCR Kit (Vircell) assay shows a good sensitivity and specificity and constitutes a promising and additional alternative to routine procedures for distinct types of clinical specimen in diagnosis STI.

Comparison of bacteriological culture method and multiplex real-time PCR for detection of mastitis

This study includes the evaluation of multiplex real-time PCR (rPCR) kit, which was developed to provide rapid diagnosis of mastitis infections, by working with milk samples of 2 different sources of mastitis and comparing the results with the classical bacteriological culture method (BC). A total of 273 bacteria were isolated in 226 samples (47.88%) out of 472 samples by BC. These were 139 (50.91%) Staphylococcus spp., 61 (22.34%) Streptococcus spp., 15 (5.49%) E. coli, 8 (2.93%) Enterococcus spp., 50 (18.31%) other bacteria. When we look at the multiplex rPCR results; 1052 positive were obtained for the gene regions of 14 different bacteria, 1 yeast, and 1 β-lactamase gene examined in 472 samples. While no searched gene region was found by rPCR in 78 (16.5%) of the 472 samples studied, at least 1 gene was detected in 394 (83.5%) samples. These 1052 positive samples by rPCR were; 263 (28.43%) Staphylococcus spp., 51 (5.51%) S. aureus, 57 (6.16%) Enterococcus spp., 49 (5.29%) C. bovis, 16 (1.73%) S. dysgalactiae, 84 (9.08%) S. agalactiae, 71 (7.67%) S. uberis, 73 (7.89%) E. coli, 14 (1.51%) Prototheca spp., 39 (4.21%) T. pyogenes/P. indolicus, 5 (0.54%) S. marcescens, 15 (1.62%) K. oxytoca/pneumonia, 117 (12.64%) Mycoplasma spp., 31 (3.35%) M. bovis, 40 (4.32%) yeast, and 127 samples (26.90%) were β-lactamase positive. When the antibiotic resistance of the isolates was evaluated, 78 (31.96%) tetracycline, 72 (29.5%) penicillin, and 60 (24.59%) clindamycin resistance were observed predominantly in Gram-positive isolates, while 6 (23.07%) tigecycline, 6 (23.07%) netilmicin, 6 (23.07%) pipercillin resistance was found in gram-negative isolates.While a bacteria and/or yeast gene was found by rPCR in 187 of 246 (76.01%) samples with no bacterial growth, a bacterium was isolated with BC in only 20 (8.84%) samples whose gene region was not found by rPCR. As a result, the multiplex rPCR system used in the diagnosis of mastitis has been found to be quite reliable as it can detect a large number of bacteria in a very short time compared to classical methods. Therefore, we advise the use of rPCR and/or culture for confirmation of clinical signs in mastitis and at routine mastitis surveillance.