Green Real-time PCR Research Articles

A sensitive batch detection of banana bunchy top virus using SYBR® Green real-time PCR

A sensitive batch detection of banana bunchy top virus using SYBR® Green real-time PCR

Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

The Effects of VEGF-A and GSTM1/GSTT1 Variants in the Susceptibility to the Chronic Rhinosinusitis with Nasal Polyposis: A Pilot Genetic Study.

Nasal polyps (NPs) are usually part of chronic rhinosinusitis with nasal polyposis (CRSwNP). However, the exact etiology of CRSwNP is still unknown. In addition, the suggested etiological causes are infection, allergy, and immunological disorders, among others, such as genetic predisposition. Moreover, it is also suggested that oxygen-free radicals play a vital role in the pathogenesis of nasal polyposis, and inflammatory cells produce free radicals during phagocytosis, which is the primary source of ROS, controlled by the glutathione S-transferase (GST) system. Although, vascular endothelial growth factor (VEGF) plays an important role in angiogenesis, it is closely interwoven with the mobilization of inflammatory cells. This pilot study evaluated the association between genetic variant VEGF-A (rs28357093) and GSTM1/GSTT1 deletion polymorphism in susceptibility to CRSwNP. A case-control study was conducted with 61 individuals diagnosed with CRSwNP and 100 healthy subjects. VEGF-A (rs28357093) and GSTM1/GSTT1 deletion polymorphisms were genotyped by RFLP-PCR and SYBR Green real-time PCR, respectively. Individuals with allergic rhinitis carriers with AC genotype (rs28357093) presented a 4-fold increased risk to CRSwNP (OR = 4.20, 95% CI = 1.31 to 13.50; p = 0.015). This evidence shows that the increased vascular permeability probably causes an inflamed nasal area leading to extensive edema and polyp growth. On the other hand, no association was verified for each genetic variant by inheritance models. Interestingly, the GSTT1 present genotype showed a protective effect on CRSwNP. In conclusion, additional studies that have larger groups in different geographic localizations may be useful to verify and assess the association between genetic variants and CRSwNP.

Comparing Typing Methods for Foot-and-Mouth Disease Virus Serotypes: Sandwich ELISA, Multiplex PCR, RT-LAMP and SYBR Green Real-time PCR

Background: Foot and Mouth Disease (FMD) poses a significant economic threat to the cattle population in Assam, with recurrent outbreaks occurring annually. Methods: This study aimed to compare different diagnostic techniques for the molecular detection and serotyping of Foot and Mouth Disease Virus (FMDV) outbreaks in the region. Epithelial tissue samples (n=29) were collected following standard procedures. SYBR Green real-time PCR targeting the 3D gene, sandwich enzyme-linked immunosorbent assay (S-ELISA), multiplex PCR (mPCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) were utilized for detection and serotyping. Results: All 29 tissue samples tested positive for FMDV using SYBR Green real-time PCR, mPCR, and RT-LAMP, with 100% congruence in serotyping (20 serotype O, 9 serotype A). In contrast, S-ELISA detected only 86.21% of samples as positive (17 serotype O, 8 serotype A). Statistical analysis revealed significant differences between the sensitivity of S-ELISA and molecular techniques (p = 2.91×10-7).The findings underscore the superior sensitivity of SYBR Green real-time PCR, mPCR, and RT-LAMP, highlighting their potential for enhancing FMDV surveillance and control efforts in Assam.

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Prevalence of Mycobacterium leprae and Mycobacterium lepromatosis in roadkill armadillos in Brazil

To evaluate the prevalence of Mycobacterium leprae and Mycobacterium lepromatosis in road killed armadillos identified along Brazilian regions, samples of liver, spleen, muscle, ear, nose and tail were collected on highways from 78 animals. The armadillos were of four different species, Cabassous tatouay, Dasypus novemcinctus, Dasypus septemcinctus and Euphractus sexcinctus. After DNA extraction from two tissues, specific primers were used for the detection of each pathogen using SYBR green qualitative Real-Time PCR, and amplicons were sequenced. The species with the highest prevalence was D. novemcinctus, mainly in the Central-West, South, and Southeast regions of Brazil. We detected M. leprae DNA in 32 (41 %) of the 78 individuals and M. lepromatosis DNA was not identified in any of the examined samples. The zoonotic component of leprosy may play a role in the transmission of the disease in endemic areas in which environmental conditions and contact with reservoirs must be investigated.

Analysis of MiR-20b, MIR-197 markers for differentiation between forensic body fluids encountered in sexual assault cases

AbstractIdentifying body fluids can be a critical clue that aids in reconstructing the crime scene. Semen and vaginal fluid identification is crucial, especially in cases of sexual assault. The majority of forensic studies focused on identifying normal body fluids and neglected the expression variation of semen in pathology. To differentiate between vaginal fluids, fertile and infertile semen samples (oligospermia and azoospermia) using miR 20b and miR197. A total of 48 body fluid samples, divided as 16 vaginal fluids, 16 fertile semen, and 16 infertile semen samples (8 with oligospermia and 8 with azoospermia), were collected, and the expression levels of miR-20b and miR-197 were detected by the SYBR Green real-time quantitative PCR technique. Our results showed significant different expression of these miRNAs in normal semen compared to vaginal and infertile semen. Moreover, we designed a model based on Fisher’s discriminant function to forecast the group affiliations of unidentified samples. With three novel equations, we were able to accurately distinguish between semen and vaginal fluid, fertile and infertile semen, and oligospermia and azoospermia semen samples with validation accuracy of 81.3%, 100%, and 100%, respectively. MiR-20b and miR-197 expression levels are efficient and appropriate markers to distinguish semen from vaginal fluid and to differentiate between fertile and infertile semen samples. However, the present study is a preliminary study based on clinical samples, and the potential role of these markers in differentiating real crime scene samples is still unknown, so we recommend further research to investigate these markers expression while using forensic samples.

Molecular investigation and genetic characterization of feline leukemia virus (FeLV) in cats referred to a veterinary teaching hospital in Northern Italy

Feline leukemia virus (FeLV) is responsible for feline leukemia syndrome in domestic cats. The prevention and control of disease caused by FeLV are primarily based on vaccination and identification and isolation of infected subjects. Antigen diagnostic methods, which are the most widely used in clinical practices, can be associated to molecular tests to characterize the FeLV detected. In this study, a quantitative SYBR Green Real-Time PCR (qPCR) assay was used to detect FeLV proviral DNA in blood samples from antigen positive cats referred to a veterinary teaching hospital in Northern Italy in 2018–2021. To genetically characterize the identified viruses, a portion of the viral envelope (env) gene was amplified using six different end-point PCRs and sequenced. Twenty-two of 26 (84.6%) cats included in the study tested positive by qPCR assay. This suggests a high performance of the qPCR adopted but further studies are required to investigate the cause of discordant results between the antigen test and qPCR in four cats. From env gene analysis, 15/22 qPCR-positive cats were infected by FeLV subtype A and 5/15 shown coinfection with subtype B.

Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp Pathogens.

Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.

Using of Real Time PCR with Propidium Mono Azide for Detection of Viable but Non Culturable Enterobacteriaceae Isolated from Raw Milk Samples

A total of 260 samples of raw milk were gathered from a variety of farms, street vendors, residences, and dairy stores in the governorate of EL-Menoufia. 220 (84.6%) of the 260 samples had Enterobacteriaceae. At species level, Klebsiella and E. coli were most predominant at 40.4% and 32.6% respectively, followed by 21.2% Citrobacter freundii and 7.3% Proteus while Salmonella showed the lowest 1.9%. Using SYBER green real-time PCR, thirty uncultured samples were subjected to rpoB genotypic identification for viable Enterobacteriaceae detection, and all thirty (100%) of the samples showed the gene. By using SYBER green real-time PCR after PMA treatment, 10 (33.3%) of these 30 culture-negative samples showed the presence of VBNC enterobacterial isolates.

Study the mRNA level of IL-27/IL-27R pathway molecules in kidney transplant rejection.

Renal transplantation stands as the sole remedy for individuals afflicted with end-stage renal diseases, and safeguarding them from transplant rejection represents a vital, life-preserving endeavor posttransplantation. In this context, the impact of cytokines, notably IL-27, assumes a critical role in managing immune responses aimed at countering rejection. Consequently, this investigation endeavors to explore the precise function of IL-27 and its associated cytokines in the context of kidney transplant rejection. The study involved the acquisition of blood samples from a cohort of participants, consisting of 61 individuals who had undergone kidney transplantation (comprising 32 nonrejected patients and 29 rejected patients), and 33 healthy controls. The expression levels of specific genes were examined using SYBR Green Real-time PCR. Additionally, the evaluation encompassed the estimation of the ROC curve, the assessment of the relationship between certain blood factors, and the construction of protein-protein interaction networks for the genes under investigation. Significant statistical differences in gene expression levels were observed between the rejected group and healthy controls, encompassing all the genes examined, except for TLR3 and TLR4 genes. Moreover, the analysis of the Area Under the Curve (AUC) revealed that IL-27, IL-27R, TNF-α, and TLR4 exhibited greater significance in discriminating between the two patient groups. These findings highlight the potential importance of IL-27, IL-27R, TNF-α, and TLR4 as key factors for distinguishing between individuals in the rejected group and those in the healthy control group. In the context of kidney rejections occurring within the specific timeframe of 2 weeks to 2 months post-transplantation, it is crucial to emphasize the significance of cytokines mRNA level, including IL-27, IL-27R, TNF-α, and TLR4, in elucidating and discerning the diverse immune system responses. The comprehensive examination of these cytokines' mRNA level assumes considerable importance in understanding the intricate mechanisms underlying kidney rejection processes during this critical period.

Can Multiplex SYBR Green Real-Time PCR Assay Serve as a Detection and Quantification Method Comparable to the TaqMan Method for SARS-CoV-2 Diagnosis?

The reopening of schools, business, and social sectors during the COVID-19 pandemic has caused a current increase in the number of COVID-19 cases and clusters all over the globe. While the COVID-19 pandemic is far from over, the reopening and resumption of all economic sectors are essential to recovering the world economy. Health experts all over the world have determined that the real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) method is the gold standard for diagnosing COVID-19 infections due to the test’s high sensitivity and specificity. During the past 3 years when WHO declared the COVID-19 pandemic, the cost of laboratory diagnosis of COVID-19 using a robust RT-qPCR assay is still considerably expensive, especially for low and middle-income countries. Therefore, numerous studies have reported optimized SYBR green methods which are more economical than the qPCR probe assay. Continuous diagnostic testing is vital to mitigate the spread of COVID-19. However, there is a question as to whether SYBR Green may serve as an excellent detection and quantification method for molecular diagnosis to perform SARS-CoV-2 screening. This review summarizes the numerous studies using SYBR Green RT-PCR to detect SARS-CoV-2. The reliability of SYBR Green qPCR assays for determining gene expression based on their performance is justified and the quality is comparable to the TaqMan method.

Development and Validation of a Real-Time PCR for the Molecular Identification of Thaumatotibia leucotreta (Lepidoptera: Tortricidae: Olethreutinae) Intercepted in Trade

The false codling moth, Thaumatotibia leucotreta (Meyrick), is a polyphagous pest indigenous to most of sub-Saharan Africa. This species is considered to be quarantined throughout much of the world, in part due to its extensive host range and significant damage caused by larval feeding. We developed a specific real-time PCR assay that allows for rapid and reliable identification of T. leucotreta. More than 150 target specimens were sequenced using an Illumina whole-genome shotgun approach to identify the most suitable loci for assay development. A hydrolysis probe that binds to a segment of the internal transcribed spacer 2 (ITS2) region was designed and used with a generic internal control targeting 18S rDNA. The assay was examined for cross-reactivity by testing additional Thaumatotibia species and representatives of related Olethreutine leafroller genera such as Cryptophlebia, Cydia, and Grapholita, which are often encountered in the same geographic region and on the same hosts as T. leucotreta. We compared our newly developed test to previously published TaqMan real-time PCR, SYBR Green real-time PCR, and loop-mediated isothermal amplification (LAMP) tests. Our newly developed real-time PCR assay outperformed all three tests in terms of analytical specificity, with 100% accuracy. These results will help to further improve diagnostic standards for molecular identification of T. leucotreta. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Establishment of a SYBR Green I-based real-time PCR for the detection of decapod iridescent virus 1

Decapod iridescent virus 1 (DIV1) is an emerging pathogen that mainly threatens decapod crustaceans, causing high mortalities and leading to huge economic losses. In this study, a pair of specific primers were designed for the major capsid protein (MCP) gene of DIV1, and a SYBR Green I-based real-time PCR method was developed. The method displayed good linearity (R2 = 1.000) and good repeatability in detecting standards of DIV1 MCP fragments ranging from 6.2 × 101 to 6.2 × 108 DNA copies/μl. Specificity analysis revealed that the real-time PCR was specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. Sensitivity analysis revealed that the real-time PCR could efficiently detect DIV1 DNA as low as 62 copies/μl within 35 cycles. In summary, the established real-time PCR provides an efficient, sensitive, and reliable detection method for DIV1.

Microsporidia in Commercially Harvested Marine Fish: A Potential Health Risk for Consumers.

Microsporidia are widely spread obligate intracellular fungal pathogens from vertebrate and invertebrate organisms, mainly transmitted by contaminated food and water. This study aims to detect the presence of major human-pathogenic microsporidia, i.e., Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi, in the gastrointestinal tract of commercially harvested marine fish from Mediterranean coast of the Comunidad Valenciana, Eastern Spain. A total of 251 fish, 138 farmed fish and 113 wild fish from commercial fishing were tested by SYBR Green real-time PCR, enabling the simultaneous detection of the four targeted species. E. intestinalis/hellem was found in 1.45% of farmed fish and 7.96% of wild fish, while Enterocytozoonidae was detected in 2.90% and 18.58% of farmed and wild fish, respectively. E. cuniculi was not detected in any of the analyzed specimens. To the authors' knowledge, this is the first report of E. intestinalis/hellem in fish, particularly in marine fish. Although the role of fish in these species' epidemiology remains unknown, this finding points out a potential public health risk linked to fish consumption. Further studies are necessary to characterize these microsporidia in fish hosts better and to elucidate their epidemiological role.

Comparison of quantitative real-time PCR targeting <i>nuc</i> gene and culture-based plate count methods for quantification of <i>Staphylococcus aureus</i> in raw cow milk

Staphylococcal food poisoning (SFP) is caused by ingestion of enterotoxins produced by enterotoxigenic Staphylococcus aureus when the cell population exceeds 5 Log CFU per gram/ml of food. The Objectives of this study were to evaluate the performance of SYBR Green 1-based quantitative real-time PCR (qPCR) targeting the nuc gene for the quantification of S. aureus in milk and to compare the assay with the plate count method. The qPCR and the plate count were applied for the quantification of S. aureus in 92 naturally contaminated and artificially spiked bulk milk samples. Optimized standard curves were generated as the qPCR employed the absolute quantification method. The qPCR assay discriminates S. aureus from other Staphylococcus species with a large difference in quantification cycle (Cq) (Mean S. aureus Cq = 13.83± 0.93; other staphylococci Cq= 30.34 ± 2.65). The standard curve showed 91 % amplification efficiency and 0.98 coefficients of correlation (R2 ). The detection and quantification limit of the assay was 18 copies of the nuc gene. The precision of the assay as expressed by standard deviation was 0.12 – 0.3 for intra-assay and 0.29 – 0.5 for inter-assay variability. In artificially contaminated milk, the R2 between CFU ml-1 and S. aureus cell equivalent (SCE) ml-1 was 0.95, which implies, the estimation of CFU ml-1 in raw milk by qPCR is possible. A statistically significant (p< 0.05) difference in S. aureus count was documented between qPCR and plate count. The average SCE (5.59 ±1.55 Log SCE ml-1) estimated by qPCR was one Log higher than CFU (4.46 ± 1.06 Log CFU ml-1) estimated by plate count. Furthermore, 28% of the samples with < 5 Log ml-1 S. aureus by plate count had > 5 Log ml-1 by qPCR. Hence, the qPCR is recommended for routine and research work for its advantage of rapid, sensitivity, and reliability. Further study on validation of the qPCR protocol in different food matrixes for quantification of foodborne pathogens and cost-benefit analysis of the assay is required.

Study of the gut microbiome in Egyptian patients with Parkinson’s Disease

BackgroundRecently, an important relationship between Parkinson’s disease and the gut microbiota, through the brain-gut axis interactions, has been established. Previous studies have declared that alterations in the gut microbiota have a great impact on the pathogenesis and clinical picture of Parkinson’s disease (PD). The present study aimed to identify the gut microbiome that is likely related to Parkinson’s disease as well as their possible relation to clinical phenotypes.MethodsThirty patients with Parkinson’s disease, who presented to the Parkinson’s disease Neurology Clinic of Alexandria University Hospital were enrolled in our study. A cross-matching control group of 35 healthy subjects of similar age and sex were included. Stool specimens were taken from each. Quantitative SYBR Green Real-Time PCR was done for the identification and quantitation of selected bacterial phyla, genera and/or species.ResultsThere was a significant increase in Bacteroides and a significant decrease of Firmicutes and Firmicutes / Bacteroidetes ratio and Bifidobacteria in PD patients. Although Prevotella was decreased among PD patients relative to the healthy control, the difference was not statistically significant. Comparing the PD clinical phenotypes with the control group, the Mixed phenotype had significantly higher Bacteroides, Tremors predominant had lower Firmicutes and Firmicutes / Bacteroidetes ratio, and both tremors and postural instability and gait disability (PIGD) phenotypes had lower Bifidobacteria. However, there was no statistically significant difference between these phenotypes. Furthermore, when comparing tremors and non-tremors predominant phenotypes; Lactobacilli showed a significant decrease in non-tremors predominant phenotypes.ConclusionsThe current study showed evidence of changes in the gut microbiome of Parkinson’s disease patients compared to the healthy controls. These observations may highlight the importance of the identification of microbiome and specific bacterial changes that can be targeted for the treatment of Parkinson’s disease.

The effect of the BK polyomavirus large T antigen on the function and maturity of the CD4+ T cell subsets in kidney transplant recipients

BackgroundIn kidney transplant recipients (KTRs) who are immunosuppressed, human BK polyomavirus (BKPyV) infection can be reactivated, resulting in BKPyV-associated nephropathy (BKPyVN). Considering that BKPyV inhibits CD4+ T cell differentiation, we investigated the effect of BKPyV large T antigen (LT-Ag) on the maturation of CD4+ T cell subsets during active BKPyV infection. MethodsIn this cross-sectional study, we examined the following groups: 1) five KTRs with active viral infection (BKPyV+ KTRs), 2) five KTRs without active viral infection (BKPyV−KTRs), and 3) five healthy controls. We measured the frequency of CD4+ T cells and their different subsets, such as naive T cells, central memory T cells (Tcm), and effector memory T cells (Tem). All these subsets were analyzed by flow cytometry in peripheral blood mononuclear cells (PBMCs) stimulated with the overlapping BKPyV LT-Ag peptide pool. In addition, CD4+ T cell subsets were analyzed by flow cytometry for the presence of CD4, CCR7, CD45RO, CD107a, and granzyme B (GB). In addition, mRNA expression of transcription factors (TFs) such as T-bet, GATA-3, STAT-3, and STAT-6 was examined. The probability of inflammation with perforin protein was examined by SYBR Green real-time PCR. ResultsAfter stimulation of PBMCs, naive T cells (CD4+CCR7+CD45RO−) (p = 0.9) and CD4+ T cells which release CD107a+ (CD4+CD107a+Geranzyme B−) (p = 0.9) T cells were more abundant in BKPyV+ KTRs than in BKPyV− KTRs. In contrast, central memory T cells (CD4+CCR7+CD45RO+) (p = 0.1) and effector memory T cells (CD4+CCR7−CD45RO+) (p = 0.1) were more abundant in BKPyV− KTRs than in BKPyV+ KTRs.The mRNA expression levels of T-bet, GATA-3, STAT-3, and STAT-6 were significantly higher (p < 0.05) in BKPyV− KTRs than in BKPyV+ KTRs which may be due to a higher differentiation level of CD4+ T cells. Due to inflammation, the mRNA expression level of perforin was higher in BKPyV+ KTRs, than in BKPyV− KTRs, but the difference was not significant (p = 0.175). ConclusionsThe high number of naive T cells after PBMC stimulation with the LT-Ag peptide pool was observed in BKPyV+ KTRs due to the interaction of LT-Ag with T cells. This means that BKPyV by using its LT-Ag can inhibit the naive T cell differentiation to other T cell subsets like central and effector memory T cells. However, the frequency of CD4+ T cell subsets and the combination of the activities of these cells with the expression profile of the target genes in this study may be efficient in treating and diagnosing BKPyV infections in kidney recipients.

Development of a novel single-tube SYBR Green real-time PCR assay for simultaneous detection of Brucella spp. and Listeria monocytogenes by melt curve analysis

Brucella spp. and Listeria monocytogenes are recognised as important food safety and public health concerns. We provide a unique single-tube real-time platform for simultaneous yet differential detection of Brucella spp. and L. monocytogenes in milk using melt curve analysis. Melting curve analysis revealed two different peaks at melting temperatures of 79.91 ± 0.56 °C and 87.51 ± 0.26 °C for L. monocytogenes and Brucella spp., respectively. The technique detected only L. monocytogenes and Brucella spp., with no different melt curves for other bacterial pathogens. The limit of detection (LoD) in spiked raw milk demonstrated that the duplex test detects the target pathogens up to 10 cfu mL−1 milk. The findings show that the newly proposed method is easy-to-use, sensitive, specific, and a viable diagnostic tool for testing a wide range of foods that are concomitantly contaminated with L. monocytogenes and Brucella spp.

Real-Time PCR based Rapid and Culture-Independent Detection of Citrobacter: ‘An Alarming State for Environment and Human Health’

The changing global scenario of environmental conditions and altered microbial community has severely deteriorated the quality of water which enforced an elevated health risks on human beings due to unavailability of safe and pristine water. Citrobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. It is an opportunistic pathogen formerly used as an indicator bacterium along with other fecal coliforms namely Escherichia coli, Klebsiella and Enterobacter, but is now known to cause several diseases in human beings. In present study we collected 135 samples from potable water and riverine-systems and confirmed the existence of Citrobacter using the conserved sequence of gnd (6-phosphogluconate dehydrogenate) gene with the application of SYBR green real-time PCR, which demonstrated extremely high specificity, fidelity and stringency in detection strategy. The Citrobacter counts in potable water were in the range of zero to 1.54 x104 ± 572.88 cfu/100ml. The Citrobacter levels in surface water were 2.18 x104 ±848, 1.54 x104 ±462, 3.53 x104 ±1299, 2.57 x104 ±912, 8.78 x103 ±201, 1.28 x104 ±370, CFU/100ml; associated with hydrophytes were 6.24 x104 ±1535, 8.42 x104 ±2711, 1.19 x105 ±4792, 1.12 x105 ±4177, 8.32 x104 ±2329, 8.08 x104 ±2149 CFU/10g and in the sediments the counts were 2.50 x104 ±647, 5.02 x104 ±1852, 3.26 x104 ±1186, 7.65 x104 ±2884, 1.50 x104 ±355, and 2.56 x104 ±737 CFU/10g at sampling site # 1, site # 2, site # 3, site # 4, site # 5 and site # 6, respectively. The present assay could be applied in the detection and regular monitoring of potable water and other environmental samples to check waterborne diseases and outbreaks caused by Citrobacter.