TaqMan Real-time Polymerase Chain Reaction Research Articles

Evaluating the effect of gargling with hydrogen peroxide and povidone-iodine on salivary viral load of SARS-CoV-2: A pilot randomized clinical trial.

This study evaluates the salivary viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in hospitalized patients and outpatients before and after gargling with 1% hydrogen peroxide and 0.25% povidone-iodine in comparison with normal saline. This clinical trial was conducted on 120 participants with laboratory-confirmed coronavirus disease 2019 (COVID-19) in two groups: outpatients (n = 60) and hospitalized patients (n = 60). In each group, the patients were randomly divided into three subgroups of 20 based on their given mouthwash for gargling (hydrogen peroxide, povidone-iodine, or normal saline). Two saliva samples were taken from each patient: the first one before gargling and the second one 10 minutes after gargling 10 ml of the respected mouthwashes for 30 seconds. The TaqMan real-time polymerase chain reaction (PCR) amplification of SARS-CoV-2 was used to measure the viral load. Saliva samples from 46% of patients were positive for coronavirus before gargling the mouthwashes. The percentage of patients with an initial positive saliva sample was significantly higher in the outpatient group (83.3%) than in the hospitalized group (5.4%) (P = 0.01). According to the findings, gargling any mouthwash similar to saline did not reduce the viral load (P > 0.05). The saliva of COVID-19 patients in the initial stage of the disease was more likely to contain SARS-CoV-2 than the saliva of the hospitalized patients. Gargling hydrogen peroxide or povidone-iodine did not reduce the salivary SARS-CoV-2 viral load.

Dual function of anti-biofilm and modulating biofilm equilibrium of orthodontic cement containing quaternary ammonium salt.

The objectives of this study were to incorporate dimethylaminohexadecyl methacrylate (DMAHDM) into resin-modified glass ionomer cement (RMGI) to develop a novel orthodontic cement which endowed RMGI with strong antibacterial ability and investigated its modulation biofilm equilibrium from cariogenic state to non-cariogenic state for the first time. Cariogenic Streptococcus mutans (S. mutans), and non-cariogenic Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) were selected to form a tri-species biofilm model. RMGI incorporated with different mass fraction of DMAHDM was examined: biofilm colony-forming units, metabolic activity, live/dead staining, lactic acid and exopolysaccharides productions. TaqMan real-time polymerase chain reaction was used to determine changes of biofilm species compositions. The results showed RMGI containing 3% DMAHDM achieved strong antibacterial ability and suppressed the cariogenic species in biofilm, modulating biofilm equilibrium from cariogenic state to non-cariogenic state tendency. The novel bioactive cement containing DMAHDM is promising in fixed orthodontic treatments and protecting tooth enamel.

Newborn screening for spinal muscular atrophy in Osaka -challenges in a Japanese pilot study-

ObjectiveThis study aimed to establish an optional newborn screening program for spinal muscular atrophy (SMA-NBS) in Osaka. MethodsA multiplex TaqMan real-time quantitative polymerase chain reaction assay was used to screen for SMA. Dried blood spot samples obtained for the optional NBS program for severe combined immunodeficiency, which covers about 50% of the newborns in Osaka, were used. To obtain informed consent, participating obstetricians provided information about the optional NBS program to all parents by giving leaflets to prospective parents and uploading the information onto the internet. We prepared a workflow so that babies that were diagnosed with SMA through the NBS could be treated immediately. ResultsFrom 1 February 2021 to 30 September 2021, 22,951 newborns were screened for SMA. All of them tested negative for survival motor neuron (SMN)1 deletion, and there were no false-positives. Based on these results, an SMA-NBS program was established in Osaka and included in the optional NBS programs run in Osaka from 1 October 2021. A positive baby was found by screening, diagnosed with SMA (the baby possessed 3 copies of the SMN2 gene and was pre-symptomatic), and treated immediately. ConclusionThe workflow of the Osaka SMA-NBS program was confirmed to be useful for babies with SMA.

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: We exposed, over 20 generations, Vgsc-L995F An. gambiae Tiassalé strain larvae 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.

Identification and Quantification of Waddlia Chondrophila in Women with Recurrent Pregnancy Loss

Background and Aims: Recurrent pregnancy loss (RPL) is one of the important complications of pregnancy. Only 50% of cases have a known cause of RPL. One of the causes of RPL is Waddlia Chondrophila (W. Chondrophila), but no information is available regarding its in Iran. This study aimed to develop a Taqman Real-Time polymerase chain reaction assay to detect recA Gene of W. Chondrophilain biological samples from women with RPL in Yazd city.
 Materials and Methods: Clinical samples of women with a history of RPL, normal childbirth, and standard strain WSU 86–1044 were provided. Primers and probes of W. Chondrophila were designed. Positive control was provided based on the PUC57 vector. Technical performance was examined using a control plasmid.
 Results: Analytical sensitivity was 5 ng/µl of control plasmid DNA. No cross-amplification was observed when testing other pathogens that can detect in human vaginitis. The Taqman Real-Time PCR assay exhibited good reproducibility. This Real-Time PCR was then applied to 100 vaginal swabs. No samples were revealed to be Waddlia positive.
 Conclusions: This new TaqMan Real-Time PCR assay represents a diagnostic tool that may be used to further study the prevalence of W. Chondrophila infection in clinical specimens.

Development and validation of TaqMan probe-based real-time polymerase chain reaction for detection of Porcine Circovirus 2

Pigs are under constant threat from many infectious diseases and post-weaning multisystemic syndrome (PWMS) is one among them. The syndrome is caused by the Porcine Circovirus 2 (PCV2) which belongs to the Circoviridae family. Because the symptoms of PCV2 infection and other porcine infectious illnesses, especially porcine reproductive and respiratory syndrome (PRRS) overlap, diagnosis of the former based on clinical indicators could be challenging. A study was conducted to develop a TaqMan real-time polymerase chain reaction (PCR) for the detection of PCV2 genotypes prevalent in Kerala. Primers and TaqMan probes based on the ORF2 nucleotide sequences of the PCV2 prevalent in Kerala were designed. On testing, it was observed that the TaqMan real-time PCR was not able to detect the PCV2genotypes prevalent in Kerala. However, the designed primers (but not the probe) were able to detect these genotypes. Hence, another TaqMan assay specific for detection of 2d was designed as that genotype was predominant in Kerala. The detection limit estimated using the cloned template was found to be 310 copies of the viral genome. The assay was more sensitive in detecting the virus compared to conventional PCR.

Insulin receptor substrate-1 gene polymorphism and lipid panel data in type 2 diabetic patients with comorbid obesity and/or essential hypertension.

Objective. The hallmarks of type 2 diabetes mellitus (T2DM) are insulin resistance (IR) and insulin receptor substrate (IRS) proteins essential for the insulin signaling. IRS-1 gene has not only been shown to be associated with T2DM, but also has indicated that it may significantly correlate with diabetic complications, such as coronary heart disease and obesity. The aim of this study was to evaluate changes of the lipid panel data in T2DM patients with comorbid obesity and/or essential hypertension in connection with the IRS-1 (rs2943640) polymorphism. Methods. The study involved 33 T2DM patients and 10 healthy individuals. The IRS-1 (rs2943640) polymorphism was genotyped using a TaqMan real-time polymerase chain reaction method. Blood serum lipid panel data were determined with commercially available kits using a Cobas 6000 analyzer. Results. Analysis of the serum lipid panel data depending on the presence of the C/A alleles of IRS-1 (rs2943640) polymorphism in T2DM patients, regardless of the presence/absence of comorbidities, showed significantly lower level of high-density lipoprotein cholesterol (HDL-C) and significantly higher level of non-HDL-C in the carriers of C allele vs. carriers of A allele. In T2DM patients with comorbid obesity and essential hypertension, proatherogenic lipid changes were found in both C and A alleles carriers. Analysis of the effect of IRS-1 (rs2943640) genotypes on serum lipid panel data in T2DM patients, regardless of the presence/absence of comorbidities, showed that the CC genotype carriers had more pronounced pro-atherogenic changes vs. carriers of СА and АА genotypes. In the comorbid course of T2DM (both in combination with obesity and obesity and essential hypertension), pro-atherogenic changes were found in the carriers of the CA genotype of IRS-1 (rs2943640) polymorphism. Conclusions. The presence of the C allele of IRS-1 (rs2943640) polymorphism in both homo-zygous and heterozygous states indicates increased risk of pro-atherogenic changes in T2DM patients with comorbid obesity and/or essential hypertension.

Effect of the GHRL gene (rs696217) polymorphism on the metabolic disorders in patients with obesity in the Ukrainian population.

Objective. Over the past four decades, the prevalence of obesity has tripled and limited genetic studies with specific SNPs have been conducted, but no investigations using ghrelin and obestatin prepropeptide (GHRL) gene have been reported in the Ukrainians population. The aim of this study was to evaluate changes in the level of metabolic hormones in the blood of obese patients in relation to the GHRL (rs696217) polymorphism. Methods. The study involved 53 obesity cases and 48 non-obesity subjects (controls). The GHRL (rs696217) polymorphism was genotyped using a TaqMan real-time polymerase chain reaction method. Blood hormones were determined with commercially available kits using a Multi-skan FC analyzer. Results. Carriers of the T allele of the GHRL (rs696217) polymorphism were statistically significantly more in patients diagnosed with obesity compared to controls indicating a genetically determined cause of obesity. We also established a significant effect of the presence of the T allele of the GHRL (rs696217) polymorphism on the decrease in the adiponectin level and the increase of resistin level in obese patients. The study of the effect of genotypes (TT, GT, GG) of the GHRL (rs696217) polymorphism on the metabolic hormone levels in the blood of obese patients did not show reliably significant differences. Conclusions. The presence of the T allele of the GHRL (rs696217) polymorphism in Ukrainian population indicates an increased risk of the obesity development regardless on the homozygous or heterozygous genotype.

Association between CX3CR1rs3732378 polymorphism and neovascular age-related macular degeneration in a sample of Algerian population.

Neovascular age-related macular degeneration (nAMD) is a progressive ocular disease, responsible for central visual loss and blindness in elderly population. Increase data demonstrate that genetic factors play an important role in pathogenesis process of this disease. The aim of this study is to investigate the association between rs3732378 polymorphism in CX3CR1 gene and nAMD in a sample of Algerian patients. This case-control study consisted of 72 patients with nAMD and 124 control subjects. DNA of participants was extracted using salting out method. Genotyping was carried out using the TaqMan real-time polymerase chain reaction method. Statistical analysis was performed by SPSS.21.0. The prevalence of the risk genotype AA was higher in the nAMD group than in control group (OR=5.02, 95% CI=1.44-17.4, P=0.011). In our sample of Algerian patients, the rs3732378 polymorphism is associated with nAMD. This result may support the role of CX3CR1 gene in the pathogenesis of nAMD.

Involvement of SYCP2L and TDRD3 gene variants on ovarian reserve and reproductive outcomes: a cross-sectional study.

Single nucleotide variants have been implicated in the response to fertility treatment and a pharmacogenomic approach may help to customize therapy based on patient genome. We aimed to evaluate the effect, individual and combined, of SYCP2L (rs2153157:G>A) and TDRD3 (rs4886238:G>A) variants on ovarian reserve, response to controlled ovarian stimulation (COS) and reproductive outcomes of women undergoing in vitro fertilization (IVF) treatment. This cross-sectional study included 149 normoovulatory women undergoing IVF. Genotyping was performed using the TaqMan real-time polymerase chain reaction method. Clinical parameters and reproductive outcomes were compared according to the genotypes of the variants studied. Considering ovarian reserve, there were no significant differences among SYCP2L or TDRD3 genotypes in terms of FSH levels or AFC; however, AMH levels were significantly different in carriers of both variants. Regarding the SYCP2L rs2153157:G>A variant, lower AMH levels were observed in women carrying an AA genotype compared to women carrying a heterozygous genotype (p=0.01). Considering the TDRD3 rs4886238:G>A variant, women carrying an AA genotype presented higher AMH levels than carriers of GG and GA genotypes (p=0.025). Nevertheless, no difference was found regarding response to COS or reproductive outcomes. Considering the combined effect of the variants, women carrying the heterozygous genotype of both variants presented statistically increased AMH levels compared to SYCP2L rs2153157 AA genotype carriers and TDRD3 rs4886238 GG genotype carriers (p=0.042). Individually and combined, the SYCP2L rs2153157 and TDRD3 rs4886238 variants have an effect on AMH level.

GHRL, LEP, LEPR genes polymorphism and their association with the metabolic syndrome in the Ukrainian population.

Objective. Many conflicting results have been obtained in the study of leptin (LEP) and leptin receptor (LEPR) gene variants that are associated with the obesity and diabetes possibly due to differences in the study populations. The aim of this study was to evaluate changes in the metabolic hormones (leptin, ghrelin, adiponectin, resistin) levels in the blood of obese patients in relation to the GHRL (rs696217), LEP (rs7799039), LEPR (rs1137100, rs1137101, rs1805094) polymorphism in Ukrainian population. Methods. The study involved 53 obesity cases and 48 non-obesity subjects (controls). The GHRL, LEP, and LEPR genes polymorphism (rs696217, rs7799039, rs1137100, rs1137101, rs1805094) was genotyped using a TaqMan real-time polymerase chain reaction method. Blood hormones (leptin, ghrelin, adiponectin, resistin) were determined with commercially available kits using a Multiskan FC analyzer. Results. The study of the effect of genotypes of the GHRL (rs696217), LEP (rs7799039), and LEPR (rs1137100, rs1805094) polymorphisms on the level of metabolic hormones (leptin, ghrelin, adiponectin, resistin) in the blood of obese patients did not show reliably significant results. Thus, the presence of the LEPR genes (rs1137101) polymorphism in the Ukrainian population indicates an increased risk of the metabolic syndrome development regardless of the homozygous or heterozygous genotype (genotypes AA, AG, GG). Conclusions. We established a significant effect of the presence of the A allele and G allele of the LEPR gene polymorphism (rs1137101) on the level of leptin, ghrelin, adiponectin, and resistin in the serum of patients diagnosed with the metabolic syndrome in the Ukrainian population.

PARP-1 genetic polymorphism associated with radiation sensitivity of non-small cell lung cancer.

About 70% of non-small cell lung cancer (NSCLC) patients require radiotherapy. However, due to the difference in radiation sensitivity, the treatment outcome may differ for the same pathology and choice of treatment. Poly (ADP-ribose) polymerase 1 (PARP-1) is a key gene responsible for DNA repair and is involved in base excision repair as well as repair of single strand break induced by ionizing radiation and oxidative damage. In order to investigate the relationship between PARP-1 gene polymorphism and radiation sensitivity in NSCLC, we collected 141 primary NSCLC patients undergoing three-dimensional conformal radiotherapy. For each case, the gross tumor volumes (GTV) before radiation and that after 40Gy radiation were measured to calculate the tumor regression rate. TaqMan real-time polymerase chain reaction was performed to genotype the single-nucleotide polymorphisms (SNPs). Genotype frequencies for PARP-1 genotypes were 14.2% for C/C, 44.7% for C/G and 41.1% for G/G. The average tumor regression rate after 40Gy radiation therapy was 35.1% ± 0.192. Tumor regression rate of mid-term RT of C/C genotype was 44.6% ± 0.170, which was higher than that of genotype C/G and G/G (32.4% ± 0.196 and 34.8% ± 0.188, respectively) with statistical significance (F = 3.169 p = 0.045). The higher tumor regression rate in patients with C/C genotype suggested that G allele was a protective factor against radiation therapy. Using the median tumor regression rate of 34%, we divided the entire cohort into two groups, and found that the frequency distribution of PARP-1 gene rs3219073 had significant difference between these two groups (p < 0.05). These results showed that PARP-1 gene polymorphism may affect patient radiation sensitivity and predict the efficacy of radiotherapy. It therefore presents an opportunity for developing new therapeutic targets to improve radiotherapy outcome.

Ovarian hyperstimulation syndrome in assisted reproductive technology programs in the ­Republic of Bashkortostan

Background. Ovarian hyperstimulation syndrome is a fatal complication of unknown etiology. In connection with the increase in the frequency of this complication, it is necessary to identify prognostically significant markers to discover the risk of its development.&#x0D; Aim. To analyze the frequency of ovarian hyperstimulation syndrome and search for the association of genes for differential growth factor 9, luteinizing hormone/horiogonadotropin receptor, and follicle-stimulating hormone receptor with different responses to ovulation stimulation.&#x0D; Material and methods. A retrospective single-center cohort study of the frequency and clinical parameters of early ovarian hyperstimulation syndrome was carried out in 147 patients, the average age was 36.5 [3338] years, who underwent an in vitro fertilization program in the period 20062021 in the Department of Assisted Reproductive Technologies of the Republican Medical Genetic Center in Ufa. The study of polymorphic loci of the genes for differential growth factor 9, luteinizing hormone/choriogonadotropin receptor, and follicle-stimulating hormone receptor was performed using TaqMan technology and real-time polymerase chain reaction. Deoxyribonucleic acid was isolated from peripheral blood samples. Statistical analysis was performed using the KruskalWallis criteria, median test, Pearson's 2 test, using the Statistica 12 software package.&#x0D; Results. 7577 procedures of assisted reproductive technologies were performed. 147 (2.3%) cases of ovarian hyperstimulation syndrome were registered, 53 (0.8%) cases of moderate and severe degree, 84 (1.3%) cases of mild degree. The *CT genotype of the rs254286 polymorphic locus of the differential growth factor 9 gene was associated with a poor ovarian response 15 (0.75); (2=4.00; p=0.02; odds ratio 3.4; 95% confidence interval 1.1310.27). The distribution of allele and genotype frequencies of the other studied genes did not differ statistically significantly in all the studied groups.&#x0D; Conclusion. The frequency of ovarian hyperstimulation syndrome in the Republic of Bashkortostan was 2.3%; the *CT genotype of the rs254286 polymorphic locus of the differential growth factor 9 gene, as well as the level of anti-Mllerian hormone, can serve as markers of a poor response and ovarian hyperstimulation to ovulation stimulation.

Initial assessment of gingival biotype as a potential source of variability in the migration, contraction and gene expression of fibroblasts

ObjectiveGingival biotype refers to the clinical classification of gingiva based on the thickness of the tissue, with thick gingival tissues more resistant to trauma and recession than the thin variant. However, to date there has never been an analysis of whether fibroblasts isolated from different biotypes possess inherent phenotypic differences. We hypothesized that gingival fibroblasts from thick and thin biotype would exhibit differences in migration, contraction and gene expression in vitro in the presence of either transforming growth factor beta one (TGF-β1) or tumor necrosis factor alpha (TNFα), two major cytokines involved in wound repair. DesignMigration was quantified using closure of scratch wound assays, contraction was assessed using attached and detached collagen lattices and extracellular matrix related gene expression using Taqman Realtime polymerase chain reaction. ResultsHuman gingival fibroblasts isolated from both biotypes showed similar rates of closure of scratch wounds, which was not influenced by the addition of TGF-β1 or TNFα. Fibroblasts from both biotypes contracted detached, but not attached, collagen gels to 50 % of their original weight although this contraction was not associated with incorporation of α-smooth muscle actin into stressfibres under any tested culture condition. Analysis of gene expression showed that POSTN, and ACTA2 mRNA levels did not significantly change, but CCN2 and COL1A2 mRNA levels were significantly higher in thick compared to thin fibroblasts in response to TGF-β1. ConclusionWhile supra-cellular factors influence the healing, esthetic outcomes and recession in thin gingival biotypes, differences in gingival fibroblast gene expression in response to growth factors may also play a role and warrants further investigation.

The prevalence and influencing factors of the oropharyngeal carriage of Haemophilus influenzae in healthy children in a high-altitude area of China: A cross-sectional study.

Haemophilus influenzae is a common commensal organism of the human upper respiratory tract and an important cause of human disease. No data on H influenzae carriage rate has been carried out on the Qinghai-Tibet Plateau of China. This study aims to present the H influenzae carriage rate and influencing factors of H influenzae in healthy children <15 years of age in Qinghai Province, an area located on the Qinghai-Tibet Plateau in China. Oropharyngeal swabs for the detection of H influenzae DNA were collected between September and October 2019. Taqman real-time polymerase chain reaction was used to detect the nucleic acids from the oropharyngeal swabs. Self-designed questionnaires were used to investigate the related information among this group of children. A number of 284 children were enrolled in this study. The carriage rate of H influenzae was 44.7%. The carriage rate in cities was 47.5%, in rural areas was 21.9%, and in pastoral areas was 52.8%. The carriage rate was found to be higher among children of minority ethnic groups than those of Han ethnicity (55.6% vs 38.1%). H influenzae carriage rate was influenced by tobacco smoke exposure (adjusted odds ratio [aOR] = 2.31, 95% CI [confidence interval]: 1.14-4.70), having siblings <5 years of age (aOR = 2.36, 95% CI: 1.21-4.59), respiratory infections during the last 30 days (aOR = 2.37, 95% CI: 1.11-5.06), and parent/guardian education level (aOR = 0.08, 95% CI: 0.02-0.27). H influenzae was highly prevalent in healthy children in Qinghai Province, especially among children of minority ethnicities and those living in pastoral areas. Tobacco smoke exposure, having siblings <5 years of age, and respiratory infections during the last 30 days were risk factors for H influenzae carriage. Parents or guardians having education levels of college or higher was a protective factor for H influenzae carriage. It is of critical importance that the government take effective measures to reduce the carriage rate and the occurrence of H influenzae related diseases in susceptible populations.

Optimization of vqPCR for Reliable Detection of Viable Candidatus Liberibacter asiaticus in Citrus

Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.

Down-regulated expression of miR-99a is associated with lymph node metastasis and predicts poor outcome in stage IB cervical squamous cell carcinoma: a case-control study.

Background Lymph node metastasis (LNM) accounts for the most important route of metastasis for cervical cancer. Yet, the status of LNM is different in patients with similar clinico-pathological variables. It has been revealed that microRNAs are widely involved in the occurrence and development of various malignancies, and the tumor-suppressive or promoting effects of microRNA-99 (miR-99) family have been previously reported. This study sought to investigate the predictive value of miR-99a for lymphogenous spread and its effect on the survival of patients with early-stage cervical squamous cell cancer (CSCC).MethodsPatients with stage IB squamous cervical cancer who were treated surgically between October 2015 and November 2018 were enrolled. A total of 21 formalin-fixed paraffin-embedded tissues of pathologically confirmed positive lymph nodes were retrieved, and an additional 21 tissues of negative lymph nodes from patients well-matched on baseline characteristics were collected as the control group. TaqMan real-time quantitative polymerase chain reaction was used to examine the expression levels of miR-99a in the samples. Differential expression levels of miR-99a were compared between the 2 groups using independent sample t-test. Furthermore, the associations between miR-99a expression level and clinico-pathological parameters of these 42 patients was evaluated by Chi-square test or Fisher’s exact-probability method, and their effects on survival were assessed using Kaplan-Meier product-limit method.ResultsThere were no significant differences in baseline clinico-pathological parameters between the 2 groups (P>0.05). The expression levels of miR-99a in the node-positive group and control group were 1.61±3.09 and 16.77±30.40, respectively (P=0.029). Downregulated expression of miR-99a was closely related to depth of invasion (DOI) and lymph-vascular space invasion (P<0.05). Univariate analysis revealed that downregulated miR-99a and deeper DOI were associated with worse 5-year disease-free survival, while multivariate analysis showed that only the expression level of miR-99a was an independent factor for disease-free survival (HR =0.120; 95% CI: 0.015–0.979; P=0.048). Patients with downregulated miR-99a tended to have more unfavorable overall survival, but the difference did not reach statistical significance.Conclusions MiR-99a plays an inhibitory role in the pathogenesis of lymph node metastasis and may serve as a novel prognostic biomarker for patients with CSCC.

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Human Immunodeficiency Virus (HIV) is a virus belonging to the family Retroviridae. HIV – 1 is found to be predominant in India and many parts of Africa. The intention of this study was to quantify the HIV Proviral Deoxyribonucleic Acid (DNA) from newly infected HIV-1 individuals. Fifty patients who were tested positive for HIV were included in this study. Proviral Ribo Nucleic Acid (RNA) was extracted by QIAmp® RNA Mini Kit (QIAGEN, Germany) method. Complementary Deoxyribo Nucleic Acid (cDNA) was synthesized by using Invitrogen Superscript III cDNA synthesis Kit (USA). This cDNA was subjected to Polymerase Chain Reaction (PCR) and Gene cloning by transformation method. The quantification of Real time PCR was done by Applied Bio-System (ABI)-Prism 7700. A linear standard curve was obtained 10 copies to 106 copies per reaction. The assay had good analytic sensitivity and linear dynamic range greater than 6 logs. From the results obtained in this study, It was concluded that Taqman Real-Time PCR Assay plays a major role in monitoring the HIV infected patients in routine diagnostics and clinical practice.

SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR.

Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions. Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR. Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%–70.8%) and 35.1% (95% CI, 29.2%–41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred. Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

Occurrence and transfer characteristics of blaCTX-M genes among Escherichia coli in anaerobic digestion systems treating swine waste

Livestock waste is a known reservoir of Escherichia coli (E. coli) carrying clinically important CTX-M-type extended-spectrum β-lactamase genes (blaCTX-M), however, the occurrence and transfer characteristics of blaCTX-M genes during anaerobic digestion (AD) remain unclear. Herein, four full-scale and two parallel lab-scale AD systems treating swine waste under ambient and mesophilic conditions were investigated by both molecular- and culture-based methods to reveal the occurrence and transfer behaviors of blaCTX-M genes during AD. Real-time TaqMan polymerase chain reaction revealed 1.3 × 104–6.8 × 105 and 3.0 × 104–7.0 × 105 copies/mL of blaCTX-M groups 1 and 9 in all feeding substrates. While AD reduced the absolute abundance of groups 1 and 9 by 0.63–2.24 and 0.08–1.30 log (P < 0.05), 5.0 × 102–4.1 × 103 and 1.1 × 104–3.5 × 104 copies/mL of groups 1 and 9 remained in the anaerobic effluent, respectively. In total, 141 blaCTX-M-carrying E. coli isolates resistant to cefotaxime were obtained from the AD reactors. Whole-genome sequencing showed that blaCTX-M-65 mainly carried by E. coli ST155 was the most frequently detected group 9 subtype in the feeding substrate; whereas blaCTX-M-14 associated with the dominant clones E. coli ST6802 and ST155 became the major subtype in AD effluent. Furthermore, blaCTX-M-14 was flanked by ΔIS26 upstream and ΔIS903B downstream. The ΔIS26-blaCTX-M-14-ΔIS903B element was mainly located on the IncHI2 plasmid in E. coli ST48 and ST6802 and also the IncFIB plasmid in ST155 in anaerobic effluent. Conjugation assays showed that the plasmids harboring blaCTX-M-14 could be successfully transferred at a frequency of 10−3–10−2 cells per recipient cell. This study revealed that blaCTX-M genes remained in both the full-scale and lab-scale AD effluents of swine waste. Thus, additional efforts should be implemented to block the discharge and spread of antibiotic resistance genes to the environment.