Sample Collection,DNA Extraction,and Library Construction Protocols of the Human Microbiome Studies in the International Human Phenome Project

The human microbiome plays a crucial role in human health.In the past decade,advances in high-throughput sequencing technologies and analytical software have significantly improved our knowledge of the human microbiome.However,most studies concerning the human microbiome did not provide repeatable protocols to guide the sample collection,handling,and processing procedures,which impedes obtaining valid and timely microbial taxonomic and functional results.This protocol provides detailed operation methods of human microbial sample collection,DNA extraction,and library construction for both the amplicon sequencing-based measurements of the microbial samples from the human nasal cavity,oral cavity,and skin,as well as the shotgun metagenomic sequencing-based measurements of the human stool samples among adult par-ticipants.This study intends to develop practical procedure standards to improve the reproducibility of microbiota profiling of human samples.

Advances in the Detection of Mycoplasma hyopneumoniae by Polymerase Chain Reaction (PCR) Technology

Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.

Guidelines for Collection of Biological Samples for Clinical and Forensic Toxicological Analysis

This article aims to review general procedures for sampling of routinely collected as well as on alternative samples that may provide additional information regarding intoxication.These approaches may be applied whenever sample collection for clinical and forensic toxicology is required and should be considered as general guidelines that must be adapted to each specific case.It is expected that this article will help toxicologists and other forensic experts to accomplish their mission,since the toxicological result is first influenced by the quality and quantity of the sample available for analysis.These guidelines were approved by the European Council of Legal Medicine.

Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions

This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation(HuiShan,Wuxi,China)AGCU Expressmarker 16(EX 16)and 22(EX22)short tandem repeat(STR)amplification kits in reduced reaction volumes using direct polymerase chain reaction(PCR)amplification workflows.The commercially available PowerPlex21(PP21)System(Promega,Wisconsin,USA),which follows similar direct workflows,was used as a reference.Anticoagulate blood applied to chemically impregnated FTATM Micro Cards(GE Healthcare UK Limited,Amersham Place,Little Chalfont,Buckinghamshire,HP79NA,UK)was used to represent a complex biological sample.Allelic concordance,first‑pass success rate,average peak heights,heterozygous peak height ratios(HPHRs),and intracolor and intercolor peak height balance were determined.In reduced volume PCR reactions,the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System.The level of performance was maintained at PCR reaction volumes,which are 40%of that recommended.The EX22 and PP21 System kits possess comparable overlapping genome coverage.This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTATM Micro Cards.Allelic concordance,first‑pass success rate,average peak heights,HPHRs,and intracolor and intercolor peak height balance were determined.A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full,half,and reduced PCR reaction volumes.The PP21 System(Promega)was used as a reference kit.Where appropriate,the distributions of data were assessed using the Shapiro‑Wilk test.For normally‑distributed data,statistics were calculated using analysis of variance(ANOVA)and for nonparametric data the Wilcoxon/Kruskal‑Wallis test was used.Statistical significance was set at P<0.05.Confidence intervals for mean values were set at 95%.On using reduced volume PCR reactions in combination with dried blood spots applied to FTA sample collection cards,both the EX16 and EX22 kits were shown to generate STR profiles of sufficient quality to allow entry into National DNA databases.The performance of both EX16 and EX22 was comparable to that of the PP21 System.This study demonstrates the successful use of the Wuxi AGCU ScienTech Incorporation EX16 and EX22 kits in reduced PCR reaction volumes with complex biological samples applied to chemically impregnated FTA sample collection cards.

Identification of Colored Dyes that are Resistant to Fading on Exposure to Ethylene Oxide;Use with Indicating FTA™Sample Collection Cards

Regulatory Standards and Forensic Communities are expressing an expectation for HID products to be certified as“DNA‑free.”Recently,“DNA‑free”status was described for HID‑related products using ethylene oxide(EtO);this gas reduces the presence of amplifiable DNA and causes minimal interference to downstream HID‑analytical methods.During sample collection,indicating cards,for example,Indicating FTA™(GE Healthcare Life Sciences,UK),are used to collect and store buccal cell DNA.These cards contain a dye which changes color on application of a colorless sample.Generating“DNA‑free”indicating cards using EtO should not impact the dyes’ability to indicate sample location or the efficacy of the card in downstream HID‑analytical methods.This study was initiated to identify alternative dyes to those currently used with sample indicating collection cards.The most promising,dyes when applied to cellulose papers exhibited a uniform color distribution and excellent sample indicating properties even when mixed with chemicals associated with FTA™.When dyed cellulose papers were exposed to EtO,ultraviolet radiation,elevated temperature,and humidity,negligible fading or discoloration was observed.The presence of these dyes on cellulose papers did not interfere with direct short tandem repeat(STR)profiling.Allelic concordance,first pass success rate,and mean peak heights were comparable to samples applied to Indicating FTA.Biological samples applied to EtO‑treated dyed cellulose papers and stored>1 month produced full STR profiles of sufficient quality to allow submission to DNA databases,confirming negligible interference from EtO treatment.These alternative sample indicating dyes resist EtO‑mediated fading while fulfilling the Forensic Community’s expectation for“DNA‑free”with negligible impact on collection card performance.