In vitro antiviral activity of stabilized chlorine dioxide containing oral care products.

OBJECTIVE: To determine the in vitro antiviral activity of oral care products containing stabilized chlorine dioxide toward infectious viruses that harbor in the oral cavity. Specfically, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, human coronavirus (HCoV) 229E, influenza A (H3N2), rhinovirus type 14, adenovirus type 5, and herpes simplex virus (HSV) type 1 and 2 were examined. METHODS: Validated in vitro suspension virucidal assays were used. Test product was mixed with the test virus for 30, 60, or 120 s, neutralized with sodium thiosulfate, serially diluted in dilution medium in a 96-well plate and incubated in a carbon dioxide incubator for 7 days. The 50% Tissue Culture Infectious Dose per milliliter was determined. RESULTS: Two rinses, one oral spray and one fluoride toothpaste showed log reduction of severe acute respiratory syndrome coronavirus-2 ranging from 1.81 to 2.98 and of influenza A from 2.58 to 4.13, respectively, within 30 s of contact time; similar results were obtained at 60 s. Further, the Ultra Sensitive rinse showed 0.19, 0.75, 1.58, 1.75, 2.66, and 3.24 log reduction of severe acute respiratory syndrome coronavirus, human coronavirus 229E, rhinovirus type 14, adenovirus type 5, and herpes simplex virus type 1 and type 2, respectively, within 30 s of contact time. CONCLUSION: Stabilized chlorine dioxide containing CloSYS® oral care products reduced the viral load of multiple viruses within 30 s. The results warrant further investigation for potential in vivo applications.

Effectiveness of a Novel Dentifrice Containing Stabilized Chlorine Dioxide, Sarkosyl, and Sodium Fluoride.

This in vitro study evaluated the effectiveness of a novel dentifrice containing stabilized chlorine dioxide, sodium lauroyl sarcosinate (sarkosyl), and sodium fluoride in enhancing enamel fluoride uptake, remineralization, pellicle cleaning and inhibiting biofilm regrowth. Remineralization was measured by fluoride uptake and surface microhardness assessment tests. Artificial stains were removed and scored based on pellicle cleaning ratio. Biofilm regrowth was measured by counting colonies on the agar plates. All studies were conducted using bovine teeth specimens. The efficacy of Toothpaste C (CloSYS anticavity toothpaste) was compared with United States Pharmacopoeia Reference Dentifrice, Toothpaste B (discontinued CloSYS anticavity toothpaste formulation) and leading commercial toothpastes. The enamel fluoride uptake and remineralization by Toothpaste C was 96.1% to 303.3% and 38.0% to 102.4% higher than the tested toothpastes, respectively. The mean pellicle cleaning ratio of Toothpaste C was similar to American Dental Association Reference Material. Toothpaste C had a significant reduction in regrowth of the oral polymicrobial biofilm compared to the control. All tested toothpastes contained 0.24% sodium fluoride. Toothpaste C exhibited significantly superior performance towards fluoride uptake and remineralization compared to the tested toothpastes. Therefore, toothpaste ingredients other than sodium fluoride accounted for the enhanced fluoride uptake and remineralization.

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

DNA profiling of azoospermic semen samples from vasectomized males by using Y-PLEX 6 amplification kit.

Post-vasectomized azoospermic semen samples (N = 6) were analyzed for short tandem repeats (STR) on the Y-chromosome by using Y-PLEX 6 and the 310 Genetic Analyzer. We have observed a wide variation in the yield of extracted DNA from 12.5-1,000 ng. This variation was attributed to the number of epithelial and/or white blood cells that are present in these azoospermic samples. DNA profiles of these vasectomized males were obtained for all six Y-STR loci, namely DYS393, DYS 19, DYS389II, DYS390, DYS391, and DYS385 amplified by using the Y-PLEX 6.

Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation.

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.

Development and validation of the Y-PLEX 5, a Y-chromosome STR genotyping system, for forensic casework.

A genotyping system, Y-PLEX 5, has been developed for use in human identification. The Y-PLEX 5 enables simultaneous amplification of five polymorphic short tandem repeat (STR) loci residing on the Y-chromosome, which are DYS389I, DYS389II, DYS439, DYS438, and DYS392. As little as 0.1 ng of template DNA can be used for analysis. The specificity of the amplification reaction enabled analysis of male DNA in a male: female DNA mixture at a ratio of 1:600. Mean stutter values ranged from 3.60-10.97%. Among the different primates investigated, the DNA from orangutan exhibited amplification at DYS438 locus and from gorilla at DYS439 and DYS438 loci. The DNA from cat, dog, and horse did not yield any amplified product. Studies on development of the genotyping system, generation and description of the allelic ladder, and validation of the multiplex PCR as per the FBI Director's Quality Assurance Standards were carried out. Y-STR allele and haplotype frequencies in two populations were generated. The data indicate that the Y-PLEX 5 genotyping system is sensitive and reliable, and can be used in human forensic and male lineage identification cases.

Utility of the Y-STR typing systems Y-PLEX 6 and Y-PLEX 5 in forensic casework and 11 Y-STR haplotype database for three major population groups in the United States.

The Y-PLEX 6 and Y-PLEX 5 systems enable analysis for 11 Y-STR loci. We present here the utility of these systems in forensic casework. A total of 188 samples, including 127 evidence samples, were analyzed using either or both of the systems. The evidence sample types included fingernail scrapings, sperm or seminal fluid, epithelial cells, blood and other tissues. The Y-STR typing systems provided useful probative results in difficult cases. A reference database for Caucasian (n = 517), African American (n = 535), and Hispanic (n = 245) population groups within the United States was generated for estimating the haplotype frequency in forensic casework. Among the individuals profiled, 311 Caucasians, 412 African Americans, and 194 Hispanics provided unique profiles in their respective population datasets. This is the first report describing the haplotype database for the set of 11 Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Linkage analysis reveals that the frequencies from forensically important autosomal loci can be multiplied with the Y-STR haplotype frequency. The results from Y-PLEX systems have been accepted in courts in the United States.

Development and validation of a multiplexed Y-chromosome STR genotyping system, Y-PLEX 6, for forensic casework.

A Y-chromosome multiplex polymerase chain reaction (PCR) amplification kit, known as Y-PLEX 6, has been developed for use in human identification. The Y-PLEX 6 kit enables simultaneous amplification of six polymorphic short tandem repeat (STR) loci located on the non-recombinant region of the human Y-chromosome. These loci are: DYS393, DYS19, DYS38911, DYS390, DYS391, and DYS385. Our studies show that as little as 0.2 ng of template DNA can be used for analysis. The specificity of the amplification reaction enabled analysis of male DNA in a male:female DNA mixture at a ratio of 1:125. Among the six Y-STR loci, the maximum mean stutter percentage was 11.9 for allele at DYS38911 locus. Attempts at amplification of DNA from various animal sources revealed that the Y-PLEX 6 primers are human specific. Details of the development of the kit, generation and description of the allelic ladders, and validation of the multiplex PCR are presented. In addition, Y-STR allele and haplotype frequencies in three populations have been investigated. The data indicate that results obtained using the Y-PLEX 6 kit are robust, sensitive, and reliable and can be used in human forensic and male lineage identification cases.

AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples.

The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 µL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation.

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues.

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.

Developmental validation of the Quantifiler Duo DNA Quantification kit for simultaneous quantification of total human and human male DNA and detection of PCR inhibitors in biological samples.

The Quantifiler Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C(T)) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/microL. In addition, the multiplex assay can detect as little as 25 pg/microL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFlSTR Amplification Kit to obtain interpretable short tandem repeat profiles.

Developmental validation of the PrepFiler Forensic DNA Extraction Kit for extraction of genomic DNA from biological samples.

The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.

Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification.

Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity. Here, we report the development of a comprehensive set of human-specific, target-specific multiplex polymerase chain reaction (PCR) assays for DNA quantitation. Using TaqMan-MGB probes, our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100-0.1 ng. Human specificity was demonstrated by the accurate detection of 0.05 and 5% human DNA from a complex source of starting templates. Target specificity was confirmed by the lack of cross-amplification among targets. A high-throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X-chromosome-based system. Background cross-amplification with DNA templates derived from 14 other species was negligible aside from the male Y assay which produced spurious amplifications from other nonhuman primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.

Human DNA quantitation using Alu element-based polymerase chain reaction.

Human forensic casework requires sensitive quantitation of human nuclear DNA from complex sources. Widely used commercially available systems detect both nonhuman and human primate DNA, often require special equipment, and have a detection limit of approximately 0.1ng. Multicopy Alu elements include recently integrated subfamilies that are present in the human genome but are largely absent from nonhuman primates. Here, we present two Alu element-based alternative methods for the rapid identification and quantitation of human DNA, inter-Alu PCR and intra-Alu PCR. Using SYBR green-based detection, the effective minimum threshold level for human DNA quantitation was 0.01ng using inter-Alu- and 0.001ng using intra-Alu-based PCR. Background cross-amplification with nonhuman DNA templates was detected at low levels using inter-Alu-based PCR, but was negligible using intra-Alu-based PCR. These Alu-based methods have several advantages over currently available systems. First, the assays are PCR based and no additional unique equipment is required. Second, the high copy number of subfamily-specific Alu repeats in the human genome makes these assays human specific within a very sensitive linear range. The introduction of these assays to forensic laboratories will undoubtedly increase the sensitivity and specificity of human DNA detection and quantitation from complex sources.

Quantitative intra-short interspersed element PCR for species-specific DNA identification.

We have designed and evaluated four assays based upon PCR amplification of short interspersed elements (SINEs) for species-specific detection and quantitation of bovine, porcine, chicken, and ruminant DNA. The need for these types of approaches has increased drastically in response to the bovine spongiform encephalopathy epidemic. Using SYBR Green-based detection, the minimum effective quantitation levels were 0.1, 0.01, 5, and 1 pg of starting DNA template using our bovine, porcine, chicken, and ruminant species-specific SINE-based PCR assays, respectively. Background cross-amplification with DNA templates derived from 14 other species was negligible. Species specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect trace quantities of species-specific DNA from mixed (complex) sources. Bovine DNA was detected at 0.005% (0.5 pg), porcine DNA was detected at 0.0005% (0.05 pg), and chicken DNA was detected at 0.05% (5 pg) in a 10-ng mixture of bovine, porcine, and chicken DNA templates. We also tested six commercially purchased meat products using these assays. The SINE-based PCR methods we report here are species-specific, are highly sensitive, and will improve the detection limits for DNA sequences derived from these species.

Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements.

We have designed and evaluated a series of class-specific (Aves), order-specific (Rodentia), and species-specific (equine, canine, feline, rat, hamster, guinea pig, and rabbit) polymerase chain reaction (PCR)-based assays for the identification and quantitation of DNA using amplification of genome-specific short and long interspersed elements. Using SYBR Green-based detection, the minimum effective quantitation levels of the assays ranged from 0.1 ng to 0.1 pg of starting DNA template. Background cross-amplification with DNA templates derived from sixteen other species was negligible prior to 30 cycles of PCR. The species-specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect known quantities of species-specific DNA from mixed (complex) sources. The 10 assays reported here will help facilitate the sensitive detection and quantitation of common domestic animal and bird species DNA from complex biomaterials.