TaqMan Real-time Polymerase Chain Reaction Research Articles

Novel antibacterial orthodontic elastomeric ligature with oral biofilm-regulatory ability to prevent enamel demineralization

ObjectivesTo synthesize a novel antibacterial orthodontic elastomeric ligature incorporating dimethylaminohexadecyl methacrylate (DMAHDM) for the first time to prevent enamel demineralization during orthodontic therapy. MethodsVarious mass fractions of DMAHDM (ranging from 0 % to 20 %) were grafted onto commercial elastomeric ligatures using an ultraviolet photochemical grafting method and were characterized. The optimal DMAHDM concentration was determined based on biocompatibility and mechanical properties, and the antibacterial efficacy was evaluated in a whole-plaque biofilm model. TaqMan real-time polymerase chain reaction and fluorescence in situ hybridization were used to assess the microbial regulatory ability of the multispecies biofilms. Furthermore, an in vitro tooth demineralization model was established to explore its preventive effects on enamel demineralization. Statistical analysis involved a one-way analysis of variance and LSD post hoc tests at a significance level of 0.05. ResultsThe elastomeric ligature containing 2 % mass fraction of DMAHDM exhibited excellent mechanical properties, favorable biocompatibility, and the most effective antibacterial ability against microorganisms, which decreased by almost two logarithms (P < 0.05). It significantly reduced the proportion of Streptococcus mutans in the multispecies plaque biofilm by 25 % at 72 h, leading to an enhanced biofilm microenvironment. Moreover, the novel elastomeric ligature demonstrated an obvious preventive effect on enamel demineralization, with an elastic modulus 30 % higher and hardness 62 % higher than those of the control group within 3 months (P < 0.05). SignificanceThe integration of DMAHDM with an elastomeric ligature holds significant promise for regulating biofilms and preventing enamel demineralization in orthodontic applications.

Association of LDLR Gene Polymorphism with the Risk of Cardiovascular Disease in End-Stage Kidney Disease Patients on Maintenance Hemodialysis

Background The low-density lipoprotein receptor (LDLR) is essential for regulating intracellular cholesterol levels. Mutations in the LDLR gene can cause a increase in LDL cholesterol levels in the blood, elevating the vulnerability to cardiovascular disease (CVD). This study evaluated the correlation between the LDLR rs688 polymorphism and CVD risk in chronic kidney disease (CKD). Materials and Methods Polymorphism in this case-control study was genotyped using the TaqMan real-time polymerase chain reaction in a cohort of 100 CKD patients (Group I) and 100 healthy controls (Group II). We examined the LDLR rs688 allele and genotype distribution in 50 CKD cases with CVD and 50 cases without CVD. Results There was a significantly greater frequency of CT variant of LDL SNP rs688 in Group I than in Group II (p = 0.006). CT and TT genotypes were significantly higher in CKD patients with CVD, with odds ratios (ORs) (95% CI) of 4.3 (1.6–11.8, p = 0.004) and 7.6 (2.3–24.8, p = 0.001), respectively. Conclusion SNP rs688 C&gt;T detection in the LDLR gene showed that CT and TT genotypes are associated with elevated CVD risk in CKD.

Novel associations between MTDH gene polymorphisms and invasive ductal breast cancer: a case–control study

ObjectiveTo reveal the contributing effects of MTDH gene SNPs in the risk of invasive ductal breast cancer (IDC).Patients and methodsA case–control study was conducted, recruiting a total of 300 cases of IDC and 565 cancer-free controls from East China. Genotyping of three single-nucleotide polymorphisms (SNPs) in the MTDH gene was performed. Genomic DNA was extracted from peripheral blood samples of patients. The three SNPs (rs1311 T > C, rs16896059 G > A, rs2449512 A > G) in the MTDH gene were selected for detection using a TaqMan real-time polymerase chain reaction assay. The association between MTDH and the risk of IDC was analyzed employing an epidemiology case–control study and a multinomial logistic regression model.ResultsAmong the three evaluated SNPs, rs1311 T > C, rs16896059 G > A, and rs2449512 A > G demonstrated a significant association with an increased risk of IDC. Furthermore, stratified analysis revealed that individuals carrying the rs1311 CC genotype, rs16896059 GA/AA genotypes, and rs2449512 GG genotype were more susceptible to developing IDC in subgroups of patients younger than 53 years, without family history of IDC, pre-menopause status, clinical stage 2, high grade, with no distant metastasis or invasion, Her2-positive type, ER positive, PR positive, and Ki67 cells less than 10%. However, carriers of the rs16896059 GA/AA genotypes and rs2449512 GG genotype had an elevate the risk of IDC in patients with tumor size larger than 2 cm, post-menopause status, clinical stage 3, with invasion, lymph node infiltration, ER negative, PR negative, Her2 negative, and Ki67 cells exceeding 10%. Compared to the reference haplotype TGA, haplotypes TAA, TAG, and TGG were significantly associated with an increased IDC risk.ConclusionIn this study, we demonstrated a significant association between MTDH gene polymorphisms and an increased risk of IDC. Moreover, our findings suggested that MTDH gene polymorphisms could serve as a potential biomarker for IDC subtyping and therapeutic selection.

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/μL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

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

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

Investigating the role of VDR gene variants in multiple sclerosis susceptibility: a case–control study in Egypt

BackgroundMultiple sclerosis (MS) is a chronic inflammatory disorder. Vitamin D has a major role in preventing inflammatory disorders as well as its role in the pathophysiology of MS. Vitamin D initiates its biological responses by binding to the nuclear vitamin D receptor (VDR). Several studies have been conducted over the last decade to investigate the relationship between VDR gene variants and the risk of MS, but the results have been inconsistent and inconclusive. The objective of this study is to investigate the association between the VDR gene variants (c.1025-49C>A) and (c.1056A>G) and MS susceptibility in a sample of the Egyptian population, and to shed light on its potential role in preventing inflammatory disorders and its impact on clinical outcomes and treatment using TaqMan Real-Time Polymerase Chain Reaction (PCR). This case-control study was conducted on 100 participants, categorized into two groups. The first group included 50 patients diagnosed with relapsing-remitting multiple sclerosis (RRMS) based on the Revised McDonald MS criteria, and the second group included 50 matched healthy participants. After collecting the blood samples, deoxyribonucleic acid (DNA) was extracted and detection of the VDR: c.1025-49C>A and VDR: c.1056A>G gene variants was done using TaqMan Real-Time PCR on all involved individuals.ResultsThe distribution of the genotypes and alleles of VDR gene variants (c.1025- 49C>A) and (c.1056A>G) did not differ significantly between MS patients and healthy participants (P>0.05 in both). ConclusionHere we show in this study that there was no association between the risk of MS and the VDR gene variants (c.1025-49C>A) and (c.1056A>G) in a group of the Egyptian population which may have impact on MS therapy and outcome.

Detection and Phylogenetic Analysis of Caprine Arthritis Encephalitis Virus Using TaqMan-based qPCR in Eastern China.

Caprine arthritis encephalitis is an infectious disease caused by the caprine arthritis encephalitis virus that infects goats, sheep, and other small ruminants. An outbreak of CAEV could be extremely harmful to the goat farming industry and could cause severe economic losses. We designed specific primers and probes for the gag gene and established a TaqMan real-time quantitative polymerase chain reaction assay. This method's correlation coefficient (R2) was >0.999, and the sensitivity of the assay to the plasmid-carried partial gag gene was approximately 10 copies/µL, 1000 times higher than that of conventional PCR. No specific fluorescence was detected for other sheep viruses. Using this method, we tested 776 asymptomatic sheep blood samples and 4 neurodegenerative sheep brain samples from six farms in eastern China, and the positivity rate was 0.77% (6/780). The gag gene was partially sequenced in the three positive samples and compared with the sequences from other representative strains in GenBank. The results revealed that all three strains belonged to the B1 subtype and were most closely related to the strains from Shanxi and Gansu, previously isolated in China, with their homology ranging from 97.7% to 98.9%. These results suggest that the designed RT-qPCR assay can be used to detect subclinical CAEV in sheep and that the virus is still present in eastern China.

A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing.

Spider mites (Acari: Tetranychidae) are polyphagous pests of economic importance in agriculture, among which the two-spotted spider mite Tetranychus urticae Koch has spread widely worldwide as an invasive species, posing a serious threat to fruit tree production in China, including Beijing. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is also a worldwide pest of fruit trees and woody ornamental plants. The cassava mite, Tetranychus truncatus Ehara, is mainly found in Asian countries, including China, Korea and Japan, and mainly affects fruit trees and agricultural crops. These three species of spider mites are widespread and serious fruit tree pests in Beijing. Rapid and accurate identification of spider mites is essential for effective pest and plant quarantine in Beijing orchard fields. The identification of spider mite species is difficult due to their limited morphological characteristics. Although the identification of insect and mite species based on PCR and real-time polymerase chain reaction TaqMan is becoming increasingly common, DNA extraction is difficult, expensive and time-consuming due to the minute size of spider mites. Therefore, the objective of this study was to establish a direct multiplex PCR method for the simultaneous identification of three common species of spider mites in orchards, A. viennensis, T. truncatus and T. urticae, to provide technical support for the differentiation of spider mite species and phytosanitary measures in orchards in Beijing. Based on the mitochondrial cytochrome c oxidase subunit I (COI) of the two-spotted spider mite and the cassava mite and the 18S gene sequence of the hawthorn spider mite as the amplification target, three pairs of specific primers were designed, and the primer concentrations were optimized to establish a direct multiplex PCR system for the rapid and accurate discrimination of the three spider mites without the need for DNA extraction and purification. The method showed a high sensitivity of 0.047ng for T. truncatus and T. urticae DNA and 0.0002ng for A. viennensis. This method eliminates the DNA extraction and sequencing procedures of spider mite samples, offers a possibility for rapid monitoring of multiple spider mites in an integrated microarray laboratory system, reducing the time and cost of leaf mite identification and quarantine monitoring in the field.

Monitoring of Benzimidazole Resistance in Botrytis cinerea Isolates from Strawberry in Korea and Development of Detection Method for Benzimidazole Resistance

&lt;i&gt;Botrytis cinerea&lt;/i&gt; is a major fungal plant pathogen that causes gray mold disease in strawberries, leading to a decrease in strawberry yield. While benzimidazole is widely used as a fungicide for controlling this disease, the increasing prevalence of resistant populations to this fungicide undermines its effectiveness. To investigate benzimidazole resistant &lt;i&gt;B. cinerea&lt;/i&gt; in South Korea, 78 strains were isolated from strawberries grown in 78 different farms in 2022, and their EC&lt;sub&gt;50&lt;/subb&gt; values for benzimidazole were examined. As a result, 64 strains exhibited resistance to benzimidazole, and experimental tests using detached strawberry leaves and the plants in a greenhouse confirmed the reduced efficacy of benzimidazole to control these strains. The benzimidazole resistant strains identified in this study possessed two types of mutations, E198A or E198V, in the &lt;i&gt;TUB2&lt;/i&gt; gene. To detect these mutations, TaqMan probes were designed, enabling rapid identification of benzimidazole resistant &lt;i&gt;B. cinerea&lt;/i&gt; in strawberry and tomato farms. This study utilizes TaqMan real-time polymerase chain reaction analysis to swiftly identify benzimidazole resistant &lt;i&gt;B. cinerea&lt;/i&gt;, thereby offering the possibility of effective disease management by identifying optimum locations and time of application.

Silica nanoparticles containing nano-silver and chlorhexidine respond to pH to suppress biofilm acids and modulate biofilms toward a non-cariogenic composition

ObjectivesDental caries is caused by acids from biofilms. pH-sensitive nanoparticle carriers could achieve improved targeted effectiveness. The objectives of this study were to develop novel mesoporous silica nanoparticles carrying nanosilver and chlorhexidine (nMS-nAg-Chx), and investigate the inhibition of biofilms as well as the modulation of biofilm to suppress acidogenic and promote benign species for the first time. MethodsnMS-nAg was synthesized via a modified sol-gel method. Carboxylate group functionalized nMS-nAg (COOH-nMS-nAg) was prepared and Chx was added via electrostatic interaction. Minimal inhibitory concentration (MIC), inhibition zone, and growth curves were evaluated. Streptococcus mutans (S. mutans), Streptococcus gordonii (S. gordonii), and Streptococcus sanguinis (S. sanguinis) formed multispecies biofilms. Metabolic activity, biofilm lactic acid, exopolysaccharides (EPS), and TaqMan real-time polymerase chain reaction (RT-PCR) were tested. Biofilm structures and biomass were observed by scanning electron microscopy (SEM) and live/dead bacteria staining. ResultsnMS-nAg-Chx possessed pH-responsive properties, where Chx release increased at lower pH. nMS-nAg-Chx showed good biocompatibility. nMS-nAg-Chx exhibited a strong antibacterial function, reducing biofilm metabolic activity and lactic acid as compared to control (p < 0.05, n = 6). Moreso, biofilm biomass was dramatically suppressed in nMS-nAg-Chx groups. In control group, there was an increasing trend of S. mutans proportion in the multispecies biofilm, with S. mutans reaching 89.1% at 72 h. In sharp contrast, in nMS-nAg-Chx group of 25 μg/mL, the ratio of S. mutans dropped to 43.7% and the proportion of S. gordonii and S. sanguinis increased from 19.8% and 10.9 to 69.8% and 56.3%, correspondingly. ConclusionpH-sensitive nMS-nAg-Chx had potent antibacterial effects and modulated biofilm toward a non-cariogenic tendency, decreasing the cariogenic species nearly halved and increasing the benign species approximately twofold. nMS-nAg-Chx is promising for applications in mouth rinse and endodontic irrigants, and as fillers in resins to prevent caries.

Implication of FSHB rs10835638 variant in endometriosis in Brazilian women.

The follicle-stimulating hormone subunit beta gene rs10835638 variant (c.-211G>T) may have detrimental effects on fertility and protective effects against endometriosis. A case-control analysis was performed, aiming to investigate the possible relationship between this variant and the development and/or progression of endometriosis. This study included 326 women with endometriosis and 482 controls without endometriosis, both confirmed by inspection of the pelvic cavity during surgery. Genotyping was performed using a TaqMan real-time polymerase chain reaction assay. Genotype and allele frequencies and genetic models were compared between the groups. The genotype and allele frequencies of the rs10835638 variant did not differ between women with and those without endometriosis. Subdividing the endometriosis group into fertile and infertile groups did not result in a significant difference in these frequencies. However, the subgroup with minimal/mild endometriosis had a higher frequency of the GT genotype than the Control Group, regardless of fertility. The T allele was significantly more common in women with minimal/mild endometriosis than in the Control Group in the recessive model. The T allele is associated with the development of minimal/mild endometriosis in Brazilian women.

The Association of TNFα -308 Polymorphism and TNFα serum level in a sample of Multiple Sclerosis Iraqi patients

Tumor necrosis factor-alpha TNF-α) is a pro-inflammatory cytokine that is involved in the pathogenesis of Multiple Sclerosis. The current study was designed to examine the association between TNF-α level and TNF-α gene polymorphisms in Multiple Sclerosis of Iraqi patients. Blood samples were collected from fifty Iraqi patients who suffered from Multiple Sclerosis (20 men and 30 women) with ages ranging between 23-54 years, and 50 healthy volunteers as a control group. The serum level of TNF-α was detected by using an Enzyme-Linked Immuno-sorbent assay (ELISA), and TNF-α-308 gene polymorphism was assessed by TaqMan Real-time Polymerase Chain Reaction (Taq-RT-PCR). The results of the estimation of TNF-α level showed high elevation in the patients’ group (4.88 ± 0.17 pg/ml) with a high significance difference (P≤0.01) as compared with the control group (2.96 ± 0.09 pg/ml). While detection of TNF-α-308 polymorphism in MS patients revealed that the wild genotype G/G was 3 (6.00%), heterogeneous genotype GA was 15 (30.00%). Homogeneous genotype AA was 32 (46.00%), while G allele frequency was 0.21 and A allele was 0.79 with significant difference (P≤ 0.005) and even as in control G/G genotype was 47 (94 %), GA genotype was 3 (6.00%), AA genotype was 0 (0.00%), G allele frequency was 0.97. A allele was 0.03 with significant difference (P≤ 0.01). The result revealed a significant difference between TNF-α-308 genotype and TNFα serum level in MS patients and control. Keywords: TNF-α-308, MS, ELISA, Taq-PCR.

Identification and Molecular Characterization of Shamonda Virus in an Aborted Goat Fetus in South Africa.

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa.

Evaluation of Viral Respiratory Pathogens Among Patients Initially Tested Negative for SARS-CoV-2

Background: A number of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses commonly circulating among vertebrates, such as influenza H1N1, respiratory syncytial virus (RSV), adenoviruses, and human coronavirus (HCoV)-229E, cause symptoms similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These viruses are important causes of cold, pneumonia, and shortness of breath in humans, which have been overlooked during the coronavirus disease 2019 (COVID-19) pandemic. Furthermore, the diagnosis of infection with these viruses mostly relies on physical examination and clinical history, despite the fact that accurate molecular diagnosis is available. Objectives: This study aimed to evaluate the presence of respiratory viruses in patients who were suspected to be infected with COVID-19 yet initially tested negative for SARS-CoV-2, as it could be beneficial in developing effective control measures and more reliable testing and surveillance of such viruses. Methods: In this study, laboratory samples of 123 patients referred to Ghaem Hospital of Mashhad, Iran, were evaluated that tested negative for SARS-CoV-2 in the initial assessment while showing the clinical symptoms of COVID-19. Initial testing for SARS-CoV-2 was carried out by the TaqMan real-time polymerase chain reaction (PCR) method using a kit approved by the Ministry of Health (Pishtaz Teb, Iran). Further analysis for the presence of 17 respiratory viruses was carried out using Genova kits based on the virus genome conserved sequences of influenza H1N1, influenza B, influenza A, SARS-CoV-2, HCoV-HKU1, HCoV-OC43, HCoV-NL63, HCoV-229E, metapneumovirus, RSV, human bocavirus 1, 2, 3, parainfluenza 1, 2, 3, and adenovirus. Results: According to the results of the present evaluations, out of 123 samples that were acquired using nasal and throat swabs and that initially tested negative for SARS-CoV-2, 8 cases of influenza A (47.1%), 1 case of parainfluenza (5.9%), 1 case of HKU1/OC-43 (5.9%), 4 cases of RSV (23.5%), 1 case of HCoV-NL63/HCoV-229-E (5.9%), and 2 cases of SARS-CoV-2 (11.8%) were detected. Conclusions: Based on the results of real-time PCR tests obtained from patients who had clinical symptoms of SARS-CoV-2 infections, it can be mentioned that due to the similar symptoms of patients with respiratory viral infections, individuals with respiratory symptoms could be examined for other viral infections in addition to SARS-CoV-2 infection, and a suitable basis for their prevalence in the community could be provided.

An Efficient Extraction Method Allowing the Genetic Evaluation of Host DNA from Samples Collected for Virus Infection Diagnosis in Viral Transport Medium.

Introduction: During the COVID-19 pandemic, an extraordinary number of nasopharyngeal secretion samples inoculated in viral transport medium (VTM) were collected and analyzed to detect SARS-CoV-2 infection. In addition to viral detection, those samples can also be a source of host genomic material, providing excellent opportunities for biobanking and research. Objective: To describe a simple, in-house-developed DNA extraction method to obtain high yield and quality genomic DNA from VTM samples for host genetic analysis and assess its relative efficiency by comparing its yield and suitability to downstream applications to two different commercial DNA extraction kits. Methods: In this study, 13 VTM samples were processed by two commercial silica-based kits and compared with an in-House-developed protocol for host DNA extraction. An additional 452 samples were processed by the in-House method. The quantity and quality of the differentially extracted DNA samples were assessed by Qubit and spectrophotometric measurements. The suitability of extracted samples for downstream applications was tested by polymerase chain reaction (PCR) amplification followed by amplicon sequencing and allelic discrimination in real-time PCR. Results: The in-House method provided greater median DNA yield (0.81 μg), being significantly different from the PureLink® method (0.14 μg, p < 0.001), but not from the QIAamp® method (0.47 μg, p = 0.980). Overall satisfactory results in DNA concentrations and purity, in addition to cost, were observed using the in-House method, whose samples were able to produce clear amplification in PCR and sequencing reads, as well as effective allelic discrimination in real-time PCR TaqMan® assay. Conclusion: The described in-House method proved to be suitable and economically viable for genomic DNA extraction from VTM samples for biobanking purposes. These results are extremely valuable for the study of the COVID-19 pandemic and other emergent infectious diseases, allowing host genetic studies to be performed in samples initially collected for diagnosis.

Phenotypic resistance to pyrethroid associated to metabolic mechanism in Vgsc-L995F-resistant Anopheles gambiae malaria mosquitoes.

Background: The indiscriminate use of insecticides in agriculture and public health lead to a selection of resistance mechanisms in malaria vectors compromising vector control tools and strategies. This study investigated the metabolic response in the Vgsc-L995F Anopheles gambiae Tiassalé resistance strain after long-term exposure of larvae and adults to deltamethrin insecticide. Methods: Vgsc-L995F An. gambiae Tiassalé strain larvae were exposed over 20 generations to deltamethrin (LS) and adults to PermaNet 2.0 (AS) and combining exposure at larvae and adult stages (LAS) and compared to unexposed (NS) group. All four groups were subjected to the standard World Health Organization (WHO) susceptibility tube tests using deltamethrin (0.05%), bendiocarb (0.1%) and malathion (5%). Vgsc-L995F/S knockdown-resistance ( kdr) mutation frequency was screened using multiplex assays based on Taqman real-time polymerase chain reaction (PCR) method. Additionally, expression levels of detoxification enzymes associated to pyrethroid resistance, including CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and glutathione S-transferase GSTe2 were measured. Results: Our results indicated that deltamethrin resistance was a response to insecticide selection pressure in LS, AS and LAS groups, while susceptibility was observed in NS group. The vectors showed varied mortality rates with bendiocarb and full susceptibility to malathion throughout the selection with LS, AS and LAS groups. Vgsc-L995F mutation stayed at high allelic frequency level in all groups with a frequency between 87% and 100%. Among the overexpressed genes, CYP6P4 gene was the most overexpressed in LS, AS and LAS groups. Conclusion: Long-term exposure of larvae and adults of Vgsc-L995F resistant- An. gambiae Tiassalé strain to deltamethrin and PermaNet 2.0 net induced resistance to deltamethrin under a significant effect of cytochromes P450 detoxification enzymes. These outcomes highlight the necessity of investigating metabolic resistance mechanisms in the target population and not solely kdr resistance mechanisms prior the implementation of vector control strategies for a better impact.

TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples.

Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference. The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, LipL32(1) and LipL32(2) qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined. The LipL32(1) qPCR assay had more diagnostic value than the LipL32(2) qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the LipL32(1) qPCR assay had 100.0% sensitivity and >75.0% specificity. The LipL32(1) qPCR assay is sensitive, specific and has the potential to be applied in future studies.

Establishment and Application of a Triplex Real-Time RT-PCR Assay for Differentiation of PEDV, PoRV, and PDCoV.

Porcine viral diarrhea is very common in clinical practice and has caused huge losses to the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important pathogens of porcine viral diarrhea. Co-infection situations among these three viruses in clinics are common, which increases the difficulty of differential diagnosis. Currently, polymerase chain reaction (PCR) is commonly used to detect pathogens. TaqMan real-time PCR is more sensitive than conventional PCR and has better specificity and accuracy. In this study, a triplex real-time RT-PCR assay based on TaqMan probes was developed for differential detection of PEDV, PoRV, and PDCoV. The triplex real-time RT-PCR assay developed in this study could not detect unrelated pathogens and showed satisfactory specificity, sensitivity, repeatability, and reproducibility with a limit of detection (LOD) of 6.0 × 101 copies/μL. Sixteen clinical samples were used to compare the results of the commercial RT-PCR kit and the triplex RT-PCR for PEDV, PoRV, and PDCoV detection, and the results were completely consistent. A total of 112 piglet diarrhea samples collected from Jiangsu province were next used to study the local prevalence of PEDV, PoRV, and PDCoV. The positive rates of PEDV, PoRV, and PDCoV detected by the triplex real-time RT-PCR were 51.79% (58/112), 59.82% (67/112), and 2.68% (3/112), respectively. The co-infections of PEDV and PoRV were frequent (26/112, 23.21%), followed by the co-infections of PDCoV and PoRV (2/112, 1.79%). This study established a useful tool for simultaneous differentiation of PEDV, PoRV, and PDCoV in practice and provided valuable information on the prevalence of these diarrhea viral pathogens in Jiangsu province.

Co-infection of COVID-19 patients with atypical bacteria: A study based in Jordan.

The aim of this work was to know the prevalence of Chlamydophila pneumoniae and Mycoplasma pneumoniae in coronavirus disease 2019 (COVID-19) patients in Jordan. Also, to assess a TaqMan real-time polymerase chain reaction (PCR) assay in detecting these two bacteria. This is a retrospective study performed over the last five months of the 2021. All nasopharyngeal specimens from COVID-19 patients were tested for C. pneumonia, and M. pneumoniae. The C. pneumoniae Pst-1 gene and M. pneumoniae P1 cytadhesin protein gene were the targets. In this study, 14 out of 175 individuals with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (8.0%) were co-infected with C. pneumoniae or M. pneumoniae. Co-infection with SARS-CoV-2 and C. pneumoniae was reported in 5 (2.9%) patients, while 9 (5.1%) patients had M. pneumoniae and SARS-CoV-2 co-infection. The mean (± std) of the correlation coefficient of the calibration curve for real-time PCR analysis was -0.993 (± 0.001) for C. pneumoniae and -0.994 (± 0.003) for M. pneumoniae. The mean amplification efficiencies of C. pneumoniae and M. Pneumoniae were 187.62% and 136.86%, respectively. In this first study based in Jordan, patients infected with COVID-19 have a low rate of atypical bacterial co-infection. However, clinicians should suspect co-infections with both common and uncommon bacteria in COVID-19 patients. Large prospective investigations are needed to give additional insight on the true prevalence of these co-infections and their impact on the clinical course of COVID-19 patients.

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

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