Single PCR Reaction Research Articles

Development of a 17-plex STR typing system for the identification of individuals and parentage testing in cattle

Accurate identification of animals and the verification of their parentage can be used to pedigree populations and support selective breeding. The International Society for Animal Genetics recommended 16 cattle STRs for individual identification and parentage testing in cattle, but no multiplex STR typing system contains these 16 STRs. Here, we develop an efficient 17-plex multiplex typing system for cattle that contains the 16 ISAG recommend STRs and a sex-determining marker. Compared to the Bovine Parenting Typing Kit (containing 11 of the 16 ISAG recommend STRs), our new typing system not only increases the number of molecular markers, but also simplifies the PCR operation and shortens the time for the typing procedure (from 4.5 h to 1 h 37 min). Profile can be generated from a single PCR reaction using as little as 1 ng of DNA. The combined probabilities of paternity exclusion CPEduo and CPEtrio were 0.999804697 and 0.999999260, respectively. These results indicate that our 17-plex typing system is a fast, sensitive and species-specific method for the identification of individuals and their parentage for cattle. The application of this system will improve the efficiency of the identification of cattle individuals and their paternity, supporting population genetic research and the selective breeding of cattle.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

Establishment and Application of Mismatch Amplification Mutation Assay-PCR for Rapid Detection and Differentiation of Duck Hepatitis A Virus-1 Attenuated Vaccine and Wild Strains.

Duck hepatitis A virus type 1 (DHAV-1) is the main pathogen causing viral hepatitis in ducks, marked by high contagion and acute mortality. Live attenuated DHAV-1 vaccines are widely used to control the disease. This study aims to develop a mismatch amplification mutation assay (MAMA)-PCR for the rapid detection and differentiation of Korean DHAV-1 wild-type strains from vaccine strains. A MAMA primer was designed to target a single nucleotide polymorphism (SNPs) at position 2276 within the VP1 gene, allowing differentiation in a single PCR reaction. The MAMA-PCR accurately identified both strains, with detection limits of 100.5 ELD50/mL and 102.3 ELD50/mL, respectively. The MAMA-PCR demonstrated specificity, showing no cross-reactivity with 12 other viral and bacterial pathogens. The MAMA-PCR was applied to 89 farms, yielding results consistent with nested-PCR and sequence determination, identifying four positive farms for DHAV-1 vaccine strains. In conclusion, this study is the first to employ the MAMA-PCR method to distinguish between DHAV-1 wild-type and vaccine strains. The developed method is rapid, simple, specific, and sensitive, thereby serving as an effective tool for clinical diagnostics in identifying and differentiating between Korean DHAV-1 wild-type and vaccine strains.

Multiplex PCR Approach for Rapid African Swine Fever Virus Genotyping.

African swine fever virus (ASFV) has been spreading through Europe, Asia, and the Caribbean after its introduction in Georgia in 2007 and, due to its particularly high mortality rate, poses a continuous threat to the pig industry. The golden standard to trace back the ASFV is whole genome sequencing, but it is a cost and time-intensive methodology. A more efficient way of tracing the virus is to amplify only specific genomic regions relevant for genotyping. This is mainly accomplished by amplifying single amplicons by PCR followed by Sanger sequencing. To reduce costs and processivity time, we evaluated a multiplex PCR based on the four primer sets routinely used for ASFV genotyping (B646L, E183L, B602L, and intergenic I73R-I329L), which was followed by Nanopore ligation-based amplicon sequencing. We show that with this protocol, we can genotype ASFV DNA originating from different biological matrices and correctly classify multiple genotypes and strains using a single PCR reaction. Further optimization of this method can be accomplished by adding or swapping the primer sets used for amplification based on the needs of a specific country or region, making it a versatile tool that can speed up the processing time and lower the costs of genotyping during ASFV outbreaks.

Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

Lack of Association Between Partial Y chromosome AZFc-gr/gr Deletions and Male Infertility

A microdeletion in the azoospermia factor (AZF) region of the human Y chromosome long arm is the second most common genetic factor causing male infertility and spermatogenetic failure. The current study aimed to evaluate the occurrence of AZFc-partial gr/gr deletions in a sample of 260 infertile azoospermia and 40 fertile men as a control group from the Kurdish community using the gel-based PCR technique. To understand whether these deletions were associated with their fertility problems. Two pairs of gr/gr primers (sY1291and sY1191) and a primer of Hetero-chromatin sY160-F were used for screening for gr/gr deletion in a single PCR reaction according to EAA and EMQN protocol. Based on the results of this study, no AZF/c gr/gr deletions were found in either the infertile or fertile participants. Therefore, this finding confirms the concept that these AZFc gr/gr partial deletions are improbable to have an important impact on infertility, and the hereditary risk associated with these partial deletions is much reduced compared to deletions that affect the entire AZFc region.

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization.

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediatedLong-readSequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.

Validation of the Microreader™ 36A ID System for forensic genetic casework and database construction

ABSTRACT Autosomal short tandem repeats (STRs) are crucial in identifying humans. Herein, a developmental validation was performed for a newly generated autosomal STR kit (Microreader™ 36A ID System). The sensitivity of this kit was fine when the input DNA dropped down to 0.02 ng/uL. Mixture analysis showed that when male:female = 200 pg: 5400 pg, full profiles of both individuals could be obtained. When inhibited substances existed, all alleles were acquired up to 2000 μM haematin, 1000 ng/μL humic acid or 250 ng/μL tannic acid. The kit exhibited a high precision level with no more than 0.29 nt standard deviation of allele size, with the accuracy (the difference of allele size between the true allele and ladder allele) no more than 0.4 nt. The total discrimination power of this system in the Chinese Han and Hui population was larger than 1–0.1 × 10−30. For the full-sibling test, the identical-by-state (IBS) score varied from 37 to 46, while it was from 21 to 28 when using the AGCU Express marker 22 STR Kit. This kit can increase the autosomal STR information of a single PCR reaction and is promising in the casework of forensic genetic as well as database construction.

Optimization and Development of an Efficient 13 X-STRs Multiplex PCR System for Paternity Testing

X-chromosomal short tandem repeats (X-STRs) markers complement autosomal STR identification systems and valuable tools in complex kinship cases. Objective: To develop a multiplex PCR system that consists of 13 X-chromosome STR markers, including GATA172D05, DXS8378, DXS6801, DXS6793, DXS6810, DXS7132, GATA31E08, DXS9902, HPRTB, DXS6789, DXS7423, DXS8377, DXS981 and sex-determining locus Amelogenin. Methods: Primer sequences of all X-STR markers were acquired from the Genome databases, and the original sequences for HPRTB, DXS6789, DXS7423, DXS8377 and DXS981 were modified to eliminate primer-dimer formation and optimize melting temperatures to increase annealing efficiency. All primer pairs were labelled with fluorescent dyes to support amplification in a multiplex PCR, and the cycling conditions for multiplex PCR were optimized. Alleles for each locus were bi-directionally sequenced to determine the exact repeat size, and alleles generated in multiplex reactions were undistinguishable from alleles produced in a single marker PCR reaction. Results: The combined power of discrimination of 13 X-STRs was 2.96 x 10-13 and 2.58 x10-8 in females and males, respectively. Conclusions: In conclusion, we have developed a 14-plex PCR system that can potentially be used for parentage testing and forensic casework studies.

Abstract 252: A single-tube approach for detection of circulating tumor DNA using customized panels

Abstract Circulating tumor DNA (ctDNA) is a promising biomarker for cancer. Applications of ctDNA analysis include screening, diagnosis, treatment selection, minimal residual disease (MRD) detection and recurrence monitoring. While some applications are best served by tumor-agnostic panels covering large regions of the genome, such large panels are not universally appropriate due to limitations in sensitivity, user-friendliness, turn-around time (TAT), and sequencing cost. One application where customized, tumor-informed panels can be preferable is detection of MRD and cancer recurrence, and multiple studies have shown that ctDNA can detect residual/recurrent disease several months earlier than routine clinical imaging. This application requires high sensitivity and in most cases will involve repeat testing of longitudinal samples collected over several months or years. In this scenario, the ideal assay characteristics include tumor-informed, personalized assay panels, with a simple, rapid workflow, and with minimal cost per assay. We have developed the Fluent LBx assay to meet these requirements. Fluent LBx analysis can be performed on-site in any appropriately equipped research or clinical laboratory, avoiding the extra cost and delay associated with external testing. Fluent LBx is a variation on SiMSen-Seq (PMID 27060140) that uses modified target and adapter primer sequences to facilitate barcoding and uniform amplification of multiple targets in a single-tube library construction protocol. Library preparation can be completed in under 1.5 hours with approximately 20 minutes of hands-on time. Using this approach, target DNA molecules are first barcoded and then amplified with indexed adaptors compatible with Illumina sequencers, in a single PCR reaction. After clean-up and sequencing, barcode information is used to compile reads from individual DNA molecules and minimize base calling errors introduced during PCR and sequencing. Here we present data demonstrating uniformity, sensitivity and specificity of Fluent LBx assays using LGC SeraCare quality control standards. Additionally, we present longitudinal clinical demonstration of residual disease detection from ctDNA collected from multiple head and neck and gastrointestinal cancer patients through longitudinal monitoring post therapeutic intervention. The Fluent LBx workflow allows rapid, inexpensive, patient-specific monitoring of MRD and recurrence in multiple cancer indications. Citation Format: Robert Meltzer, Jessalyn Kaur, Ekaterina Kintsurashvili, Christopher D'Amato, Tony Godfrey. A single-tube approach for detection of circulating tumor DNA using customized panels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 252.

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Using Single Multiplex PCR Reaction to Identify Candida Species in Vaginal isolates: Comparison between Phenotypic and Molecular Methods

Using Single Multiplex PCR Reaction to Identify Candida Species in Vaginal isolates: Comparison between Phenotypic and Molecular Methods

"Biological identikit": Development of a SNPs-panel for the analysis of forensic DNA phenotyping and ancestry

Personal identification in mass disasters and in crimes is essential for humanitarian, ethical and legal reasons. In these contexts, when individuals cannot be identified by standard forensic DNA analysis, the Forensic DNA Phenotyping and the analysis of the biogeographical ancestry could help. The aim of this study was to evaluate the potential of a new panel of 891 SNPs in predicting phenotypic traits and biogeographical origin to create a “biological identikit”. In addition to fresh biological material, old evidence found at the crime scene or extracted and long-term stored DNA were tested with 41 SNPs for phenotyping and 850 SNPs for ancestry. All the SNPs were successfully incorporated into a single two-step multiplex PCR reaction using the IonAmpliSeq ™ Library Plus and applied for massive parallel sequencing with the Ion S5 platform using up to 0.05 ng/µL of DNA. The analysis of the results was carried out with an in-house predictive algorithm and consulting 20 population databases. By comparing the results obtained with identikit or video-photographic surveys, it was possible to predict phenotype and ancestry with an accuracy greater than 90%. While these new markers cannot identify a specific individual, they can be a valuable investigative tool.

A method for quick and efficient identification of cichlid species by high resolution DNA melting analysis of minibarcodes

Freshwater bodies are key in supporting aquatic and terrestrial life. Ecological balance of freshwater habitats is very vulnerable, hence, often significantly disrupted by climatic changes and anthropogenic acts. In Israel, due to its relatively arid climate, many freshwater resources have been disrupted and still are under great pressure. The Sea of Galilee is the largest surface freshwater body in the Middle East and a habitat to unique populations of several fishes, including six cichlid species. Studies on the ecology of these fish and their conservation require effective monitoring tools. In this study, a simple and efficient molecular method was developed to identify the species of these lake cichlids using high resolution melting analysis of mini DNA barcodes. The species of an individual sample can be identified by a single tube PCR reaction. This assay successfully identified sequence differences both among and within species. Here, this method identified the species for 279 small cichlid fry that could not be morphologically identified, allowing to estimate relative species abundance and map their distribution in time and location. The results are key to understand not only the ecology of young stages but also their recruitment potential to adult fish populations and their sustainability. This method can be readily implemented in further ecological studies and surveys related to these species, in the lake and its surroundings, as a tool to enhance understanding and protection of these species.

Molecular Detection of Mycoplasma Gallisepticum and Mycoplasma Synoviae Infection in Poultry

In Pakistan, production of poultry has been evolved as a good alternate of beef and mutton. In this study multiplex PCR approaches were adopted to differentiate and detect avian mycoplasma in a single PCR reaction as a step forward in the economization of PCR detection. From the total of 25 samples, 25 (100%) had a positive Mycoplasma isolation result. The biochemical test yielded the highest number of isolations, with MG accounting for 10/25. Commercial DNA-based PCR kits have been developed as a result of recent advancements in diagnostic techniques for Mycoplasma infections. Tetracyclines, fluoroquinolones, tilmicosin, tylosin, spiramycin Infections should be reduced as much as possible.

Abstract 534: An ultrasensitive high-plex assay detecting 24 PIK3CA mutations using SAGAsafe® technology and 6-color Crystal Digital PCR™

Abstract Starting from a minimally invasive liquid biopsy sample such as a blood sample, one can determine genetic and biological properties of cancer by measuring circulating tumor DNA (ctDNA). ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. The focus of this study was to demonstrate the performance of the patented SAGAsafe digital PCR chemistry using 6-color Crystal Digital PCR, by adapting a SAGAsafe assay for a clinically relevant oncogene, PIK3CA. PIK3CA encodes the p110-alpha kinase and is the target of a recently approved drug, alpelisib, for metastatic breast cancer. By combining the power of the SAGAsafe technology with that of 6-color Crystal Digital PCR on the naica system®, we present a highly multiplexed, ultrasensitive proof-of-concept PIK3CA detection assay capable of detecting and quantifying 24 PIK3CA mutations in a single digital PCR reaction. By evaluating the sensitivity, specificity and precision of the high-plex 6-color PIK3CA digital PCR assay, we show that despite the complexity of the assay, the data were extremely clean resulting in highly accurate quantifications down to mutant allele frequencies (MAFs) in the 0.01% range. In addition, superb PIK3CA mutation detection specificity and sensitivity were observed with Limits of Blank ranging from 0.003% to <0.001% MAF, and Limits of Detection ranging from 0.009% to 0.002% MAF in a high background of wild-type DNA. Moreover, a high concordance was observed between Crystal Digital PCR PIK3CA results and NGS data on breast cancer biopsies. By joining 6-color Crystal Digital PCR and ultrasensitive SAGAsafe technology, high sensitivity and specificity can be maintained even in a complex multiplex digital PCR assay. This proof-of-concept assay sets the stage for the future development of ultrasensitive, highly multiplexed cancer detection panels to achieve better patient stratification and monitoring during therapy. Citation Format: Miguel Alcaide, Anthony M. George, Yilun Chen, Cécile Jovelet, Sahbi Ben Kilani, Lao H. Saal, Allison C. Mallory. An ultrasensitive high-plex assay detecting 24 PIK3CA mutations using SAGAsafe® technology and 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 534.

Two fingerprinting sets for Humulus lupulus based on KASP and microsatellite markers.

Verification of clonal identity of hop (Humulus lupulus L.) cultivars within breeding programs and germplasm collections is vital to conserving genetic resources. Accurate and economic DNA-based tools are needed in dioecious hop to confirm identity and parentage, neither of which can be reliably determined from morphological observations. In this study, we developed two fingerprinting sets for hop: a 9-SSR fingerprinting set containing high-core repeats that can be run in a single PCR reaction and a kompetitive allele specific PCR (KASP) assay of 25 single nucleotide polymorphisms (SNPs). The SSR set contains a sex-linked primer pair, HI-AGA7, that was used to genotype 629 hop accessions from the US Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR), the USDA Forage Seed and Cereal Research (FSCR), and the University of Nebraska-Lincoln (UNL) collections. The SSR set identified unique genotypes except for 89 sets of synonymous samples. These synonyms included: cultivars with different designations, the same cultivars from different sources, heat-treated clones, and clonal variants. Population structure analysis clustered accessions into wild North American (WNA) and cultivated groups. Diversity was slightly higher in the cultivated samples due to larger sample size. Parentage and sib-ship analyses were used to identify true-to-type cultivars. The HI-AGA7 marker generated two male- and nine female-specific alleles among the cultivated and WNA samples. The SSR and KASP fingerprinting sets were compared in 190 samples consisting of cultivated and WNA accession for their ability to confirm identity and assess diversity and population structure. The SSR fingerprinting set distinguished cultivars, selections and WNA accessions while the KASP assays were unable to distinguish the WNA samples and had lower diversity estimates than the SSR set. Both fingerprinting sets are valuable tools for identity confirmation and parentage analysis in hop for different purposes. The 9-SSR assay is cost efficient when genotyping a small number of wild and cultivated hop samples (<96) while the KASP assay is easy to interpret and cost efficient for genotyping a large number of cultivated samples (multiples of 96).

Evaluation of process limit of detection and quantification variation of SARS-CoV-2 RT-qPCR and RT-dPCR assays for wastewater surveillance

Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 × 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.

Systematic Evaluation of a Novel 6-dye Direct and Multiplex PCR-CE-Based InDel Typing System for Forensic Purposes.

Insertion/deletion (InDel) polymorphisms, combined desirable characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are considerable potential in the fields of forensic practices and population genetics. However, most commercial InDel kits designed based on non-Asians limited extensive forensic applications in East Asian (EAS) populations. Recently, a novel 6-dye direct and multiplex PCR-CE-based typing system was designed on the basis of genome-wide EAS population data, which could amplify 60 molecular genetic markers, consisting of 57 autosomal InDels (A-InDels), 2 Y-chromosomal InDels (Y-InDels), and Amelogenin in a single PCR reaction and detect by capillary electrophoresis, simultaneously. In the present study, the DNA profiles of 279 unrelated individuals from the Hainan Li group were generated by the novel typing system. In addition, we collected two A-InDel sets to evaluate the forensic performances of the novel system in the 1,000 Genomes Project (1KG) populations and Hainan Li group. For the Universal A-InDel set (UAIS, containing 44 A-InDels) the cumulative power of discrimination (CPD) ranged from 1–1.03 × 10–14 to 1–1.27 × 10–18, and the cumulative power of exclusion (CPE) varied from 0.993634 to 0.999908 in the 1KG populations. For the East Asia-based A-InDel set (EAIS, containing 57 A-InDels) the CPD spanned from 1–1.32 × 10–23 to 1–9.42 × 10–24, and the CPE ranged from 0.999965 to 0.999997. In the Hainan Li group, the average heterozygote (He) was 0.4666 (0.2366–0.5448), and the polymorphism information content (PIC) spanned from 0.2116 to 0.3750 (mean PIC: 0.3563 ± 0.0291). In total, the CPD and CPE of 57 A-InDels were 1–1.32 × 10–23 and 0.999965, respectively. Consequently, the novel 6-dye direct and multiplex PCR-CE-based typing system could be considered as the reliable and robust tool for human identification and intercontinental population differentiation, and supplied additional information for kinship analysis in the 1KG populations and Hainan Li group.

A duplex PCR assay for authentication of Ocimum basilicum L. and Ocimum tenuiflorum L in Tulsi churna

The primary requirement for any valuable herbal product is an authentic plant species used in its formulation. In this study, we have attempted to develop a simple and cost effective PCR method for the identification of Ocimum basilicum and Ocimum tenuiflorum. For this purpose, we developed species-specific primers and PCR assays. The primers were designed from the chloroplast genome sequences, PCR methods were optimized and further tested for specificity and sensitivity. Methods have been validated using simulated blended materials to mimic the possible admixtures. Primers of both the species are found to be specific and sensitive enough to amplify 0.1 ng of DNA for O. basilicum whereas 0.5 ng of O. tenuiflorum. Duplex PCR assay is also optimized where, one can detect both the species in a single PCR reaction. The sensitivity of primers was further improved up to approximately 10–100 times with digital PCR assay. Chromatographic methods based on conventional chemical makers of Ocimum have also been investigated. Market samples labelled as ‘Tulsi powder’ were tested using PCR assay and chromatographic methods. Ursolic acid and oleanolic acid was detected in all the market samples whereas eugenol/methyleugenol has been detected inconsistently in the market samples. A comparison of results from molecular and chromatographic authentication methods suggested that both methods should be used in analysis to achieve maximum fidelity for the samples where there is a doubt. However, for the larger sample size, a rapid and cost-effective viable strategy is required. A duplex PCR assay developed in the current study is enough for the authentication of two Ocimum species, O. basilicum and O. tenuiflorum.