Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

Detection and quantification of Bacillus cereus and its spores in raw milk by qPCR, and distinguish Bacillus cereus from other bacteria of the genus Bacillus

Introduction The raw milk is the basic raw material of dairy products, Bacillus cereus is a typical conditional pathogenic bacteria and cold-phagocytic spoilage bacteria in raw milk. This study established a qPCR method for detecting B. cereus in raw milk Materials and Methods In this study, a qPCR method for detecting B. cereus in raw milk was established. The specificity of the method was verified by using other Bacillus bacteria and pathogenic bacteria, the sensitivity of the method was evaluated by preparing recombinant plasmids and simulated contaminated samples, and the applicability of the method was verified by using pure spore DNA. The actual sample detection was completed by using the established qPCR method Results The qPCR established in this study can specifically detect B. cereus in raw milk. The LOD of the method was as low as 200 CFU/mL, and the LOQ ranged from 2 × 10 2 to 2 × 10 8 CFU/ml, the amplification efficiency of qPCR was 96.6% Conclusins The method established in this study can distinguish B. cereus from other Bacillus bacteria, and spore DNA can be used as the detection object. This method has the advantages of strong specificity, high sensitivity, wide application range and short detection time, which is expected to be applied in the dairy industry.

CYP2D6 Genotyping for Personalized Therapy of Tamoxifen in Indonesian Women with ER+ Breast Cancer

PurposeTamoxifen, common adjuvant therapy prescribed in estrogen receptor positive (ER+) breast cancer, is metabolized by CYP2D6 enzyme into endoxifen. The phenotypes of CYP2D6, a highly polymorphic gene, vary from ultrarapid (UM), normal (NM), intermediate (IM), and poor metabolizers (PM). Studies showed that reduced CYP2D6 activity in IMs and PMs resulted in lower endoxifen level, thereby reducing therapy efficacy. This study aims to observe the distribution of CYP2D6 profiles and their corresponding endoxifen levels in Indonesian ER+ breast cancer patients.Methods151 patients who have received tamoxifen therapy for ≥8 weeks were recruited prospectively. DNA and blood samples were collected with buccal swab and finger-prick methods, respectively. Genotyping was performed using the qPCR method while metabolites measurement was performed using HPLC-tandem MS. Patients with IM/PM CYP2D6 profile were advised to increase their tamoxifen dose or switch to aromatase inhibitor, while patients with UM or NM CYP2D6 profile remained on 20 mg daily dose. Tamoxifen metabolites levels of those given 40 mg/day of tamoxifen were measured eight weeks post dose adjustment.ResultsWe found that 40.7% of patients recruited were IM. CYP2D6*10 was the most abundant allele (28.8%) and *10/*36 was the most frequently observed diplotype (23.6%). Endoxifen levels between the NM-PM, NM-IM, and IM-PM were statistically significant, and dose increase of tamoxifen successfully increased endoxifen levels in IMs to a similar level with NMs at baseline.ConclusionIndonesian women have a relatively high proportion of IMs. The correlation between CYP2D6 genotype and phenotype was shown in the significant difference in endoxifen levels among NMs, IMs, and PMs. Dose adjustment of tamoxifen to 40 mg daily positively increased endoxifen levels in IMs to a similar level as NMs. Implementing pharmacogenomics testing of CYP2D6 on ER+ breast cancer women taking tamoxifen can potentially increase the likelihood of achieving better treatment efficacy.Trial RegistrationThe trial was retrospectively registered at ClinicalTrials.gov on 18 March 2020 with identifier NCT04312347 (accessible at: https://clinicaltrials.gov/ct2/show/NCT04312347).

Comparative evaluation of cost effective extraction free molecular technique for detection of SARS-CoV-2 with reference to standard VTM based RT-qPCR method

Background and Objectives: The entire globe is undergoing an unprecedented challenge of COVID-19. Considering the need of rapid and accurate diagnostic tests for SARS-CoV-2, this study was planned to evaluate the cost effective extraction free RT-PCR technique in comparison to the standard VTM based RT-qPCR method. Materials and Methods: Paired swabs from nasopharynx and oropharynx were collected for SARS-CoV-2 testing, from 211 adult patients (≥18 years) in VTM and plain sterile tubes (dry swabs). These samples were processed and RT-qPCR was carried out as per standard protocols. Results: 54.5% of the patients were females and 45.5% were males with sex ratio 1:1.19 (M: F). 38.86% were symptomatic, of which fever (86.59%), cough (79.23%) and breathlessness (46.34%) were the most common symptoms. The positivity by VTM based method and index method was 31.27% and 13.27% respectively. Of the 27 inconclusive results from index method, 37.04% were positive, 48.15% were negative by VTM based method. However, in 40 inconclusive results by VTM based method, 90% were negative and rest remained inconclusive by index method. The sensitivity and specificity of the index method were 39.39% and 85.71% respectively. The overall agreement between VTM based method and index method was 49.59% with estimated Kappa value of 0.19. Conclusion: VTM based method showed higher sensitivity compared to the index method. The higher positivity by VTM based method, suggests that VTM based method could plausibly be a better detection method of SARS-CoV-2. Still, the index method might add value in a resource limited setups for detection of SARS-CoV-2.

Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp.

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20–60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.

A Novel qPCR Method for the Detection of Lactic Acid Bacteria in Fermented Milk

The number of live lactic acid bacteria (LAB) is an important quality indicator for yogurt, the quantitative testing of LAB has become an important task in the evaluation of product quality and function. By analyzing and comparing the performance of 16S rRNA gene and tuf gene used in absolute quantification, the tuf gene with copy number 1 was selected as the target gene of six LAB. By drawing a standard curve to achieve qualitative and quantitative detection of six strains of LAB, the detection range was found to be 1 × 103–1 × 108 copies/µL. The traditional plate colony count and Flow Cytometry (FCM) were compared with the method of qPCR, which was used in this experiment. Meanwhile, the confocal laser microscope combined with STYO 9 and propidium iodide dyes was used to determine that the content of viable bacteria in the yogurt was more than 90%, which proved that the detection result using qPCR method was closer to the true level of LAB in yogurt. Compared with the existing methods, the method in this study allowed the qualitative and quantitative detection of the six kinds of LAB in yogurt, and the distribution of live and dead bacteria in yogurt could be calculated.

Comparison of Telomere Length Between Buccal Cells and Blood Cells

Background Telomere length (TL) in blood has been extensively studied as a biomarker of aging and aging-associated disease. TL in blood cells is commonly used as a proxy for TL in other tissue types. The source of DNA of adequate quality and quantity is an important consideration in telomere length analysis. Compared to blood cells, buccal cells easy for genomic DNA preparation would facilitate the rapid and reliable telomere length analysis. However, the feasibility of buccal cells for TL analysis remains yet unestablished. Methods A total of 52 participants ranged in age from 18 to 80 years including 24 males and 28 females were included in this study. Both buccal and blood samples were taken at the same time by using buccal cell swabs and fingertip stick from each participant. Relative telomere length (RTL) was analyzed using the quantitative real-time polymerase chain reaction (qPCR) method. Results The results indicate that there is a strong positive correlation between buccal RTL and blood RTL and negative correlation between both buccal RTL and blood RTL with age. Conclusion The validity of sampling using buccal cell swabs provides simple operation and good reproducibility for telomere length analysis, which overcomes the discomfort and risk of infection caused by blood sampling.

Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.

Investigation of Cumulus Cell Factors Associated With Embryo Development and Pregnancy Outcome in Human IVF

<p>Recent evidence suggests that the expression of some candidate genes in cumulus cells (CC) have the potential to serve as markers of oocyte quality. The aims of this study were: 1) to validate a multiplex quantitative polymerase chain reaction (QPCR) method to measure four genes simultaneously in extracts from individual CC and; 2) to investigate the relationships in individual cumulus-oocyte-complexes (COC) amongst the expression levels of a range of candidate genes (N=8) from individual CC, the numbers of CC per COC and developmental indicators (good blastocyst development and live birth outcome) of the associated oocytes following in vitro fertilization (IVF) with intra-cytoplasmic sperm injection (ICSI). Sixty eight women were recruited for this study following approval from NZ Multi-Regional Ethics Committee and classified into four research groups: young and healthy women (<38 years, N=25), young women with polycystic ovarian syndrome (<38 years, N=11), young with diminished ovarian reserve (<38 years, N=12) and older and healthy women (≥40 years, N=20). Following exogenous rFSH-assisted ovarian stimulation, 608 COC were collected and subjected to ICSI. Oocyte and embryo quality, and pregnancy outcomes were recorded. Expression levels of the following candidate genes HAS2, FSHR, SLC2A4 (GLUT4), ALCAM, SFRP2, VCAN, NRP1 and PR in CC extracts from individual COC were measured by TaqMan QPCR and normalized against the house-keeping gene, RPL19. The numbers of CC from individual COC were calculated from RPL19 expression levels plotted against a standard curve of CC number. These results were then assessed against the outcomes of the associated oocytes following ICSI. HAS2, FSHR, ALCAM, VCAN, NRP1 and PR mRNA were detectable in most samples (98.5%) whereas those for SLC2A4 and SFRP2 were generally undetectable. The minimum number of CC required for QPCR was estimated to be ~70. The mean levels of FSHR mRNA were up-regulated in young women with PCOS compared to those in the other two groups of women <38y. Expression levels of HAS2 across all four groups of women were correlated to both biological (age, basal serum FSH and serum AMH) and treatment (amount of rFSH used for stimulation, follicle numbers and COC retrieved) variables. Investigations related to oocyte development in young and healthy women showed that 1) mean mRNA levels of VCAN, HAS2 and PR were higher (P=0.002, P<0.05, P<0.05, respectively) in CC associated with oocytes that resulted in good quality blastocysts and those of VCAN were higher (P<0.05) in CC associated with oocytes that resulted in a live birth, compared with those with developmental failure. However, the expression levels of all measurable candidate genes were highly variable for individual CC from COC from each woman. Indeed, 99.7% of individual COC were different in CC mRNA levels and cell composition. The application of a ranking method to score the relative CC mRNA levels of selected candidate genes from each COC recovered from individual women was evaluated. This approach demonstrated a predictive power of 80% efficiency for selecting good quality oocytes (at least one), whilst requiring the insemination of no more than three oocytes in any treatment cycle. Furthermore, this selection method resulted in a pregnancy and live birth rate of 60 and 52% respectively (N=25 women). This outcome is similar to that achieved when all metaphase II (MII) oocytes are inseminated. In conclusion, this study has validated a multiplex QPCR method to quantify the expression levels of four genes in CC of individual human COC simultaneously using as few as 70 cells. Moreover, that there is sufficient cDNA so that many more candidate genes can be measured in the same extract. From the knowledge of the mRNA levels of at least four genes, VCAN, FSHR, HAS2 and PR in CC, it is possible to improve upon existing biological indicators the potential to predict good blastocyst formation and a successful pregnancy outcome. It is concluded that the application of a multiplex QPCR approach to assess the expression levels of a limited number of marker genes in CC can be used to select the best oocytes for successful pregnancy outcomes.</p>