Psychedelic fungus (Psilocybe sp.) authentication in a case of illegal drug traffic: sporological,molecular analysis and identification of the psychoactive substance

In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study.Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves.This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1?μm and width 6.4?μm. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.     

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples*†

Abstract: Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C‐stretch and CA repeat. To observe which CA “alleles” were present in each tissue, PCR products were cloned and re‐sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.     

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60‐year‐old and 400–500‐year‐old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.     

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

脱落毛发线粒体DNA HV1区序列测定的研究

目的　对脱落毛发线粒体DNAHV1区序列测定方法进行研究。方法　嵌合扩增结合末端荧光标记DNA测序。结果　对 2 0例脱落毛发进行分析获得了明确的测序结果 ,与来自同一个体的血液所测得的DNA序列进行比较 ,完全相同。结论　嵌合扩增在对脱落毛发进行线粒体DNA多变区序列分析中是一种有效的方法 ,在法医DNA检验中具有实用价值。     

Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs

A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.     

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies

We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.     

Forensic casework analysis using the HVI/HVII mtDNA linear array assay

The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.     

Heteroplasmies detected in an amplified mitochondrial DNA control region from a small amount of template

When mitochondrial DNA (mtDNA) heteroplasmies are detected, they often confound forensic identification, especially if they are the result of poor biological sampling. In this study, we determined the ratio of heteroplasmy in samples that were amplified from a very small amount of template mtDNA or a few cells using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis. As a result, more than half of the detected sequences (i.e., 17/20, 15/20, and 14/20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. Additionally, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These results indicate the risks associated with using highly sensitive mtDNA techniques in forensic investigations because of the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample.     

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.     

Mitochondrial DNA typing screens with control region and coding region SNPs

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.     

Fluorescent SSCP of overlapping fragments (FSSCP-OF): a highly sensitive method for the screening of mitochondrial DNA variation.

The mtDNA analysis (mtDNA) is increasingly being demanded for forensic purposes due to the fact that many times the use of standard nuclear marker fails to analyze degraded samples (such as bones) and specially for the analysis of hair shafts (a common sample in the crime scene). However, analysis of mtDNA sequencing implies a great lab effort when a high number of samples must be analyzed.The present work introduces a novel and reliable method for the screening of mtDNA variation in the first and second hypervariables (HV1 and HV2) regions which we have denominated fluorescent single strand conformation polymorphism (SSCP) of overlapping fragments (FSSCP-OF). FSSCP-OF is based on the basic theory of SSCP analysis and combines two complementary strategies: the use of PCR amplified overlapping fragments and fluorescent detection technology. The overlap region contains a high percentage (50%) of the d-loop mtDNA variation and for this reason, the probability to detect a polymorphic position by SSCP analysis is clearly increased in comparison to conventional SSCP methods due to the fact that the same polymorphic position is usually placed in a different "relative" position in the two overlapped fragments. The use of multicolor fluorescent technology allows also the multiplex amplification of overlapping fragment and its subsequent analysis in an automatic sequencer. We have analyzed 50 samples of unrelated individuals through the FSSCP-OF technique and we have found that using this methodology the probability to distinguish two samples with different sequences is close to 100%. FSSCP-OF has other important advantages with respect to previous screening methods, such as the automation and standardization of the protocols, which is of special interest for the forensic routine.     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy

Mitochondrial DNA (mtDNA) analysis of highly degraded skeletal remains is often used for forensic identification due largely to the high genome copy number per cell. Literature from the "ancient DNA" field has shown that highly degraded samples contain populations of intact DNA molecules that are severely restricted in size (1-4). Hand et al. have demonstrated the targeting and preferential amplification of authentic human DNA sequences with small amplicon products of 150 bp or less (1,2). Given this understanding of ancient DNA preservation and amplification, we report an improved approach to forensic mtDNA analysis of hypervariable regions 1 and 2 (HV1/HV2) in highly degraded specimens. This "mini-primer set" (MPS) amplification strategy consists of four overlapping products that span each of the HV regions and range from 126 to 170 bp, with an average size of 141 bp. For this study, 11 extracts representing a range of sample quality were prepared from nonprobative forensic specimens. We demonstrate a significant increase in MPS amplification success when compared to testing methods using approximately 250 bp amplicons. Further, 16 of 17 independent amplifications previously "unreported" due to mixed sequences provided potentially reportable sequence data from a single, authentic template with MPS testing.     

Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair

The phenomenon known as heteroplasmy can be operationally observed in some human mitochondrial DNA (mtDNA) samples. Typically, heteroplasmy manifests itself in an individual presenting two mtDNA species that differ at a single base. Heteroplasmy at two, and even possibly three sites, also may occur, but at very low rates. A recent report (Grzybowski, 2000, see ref. [13]) suggests that much higher levels of mtDNA (point substitution) heteroplasmy can occur in hair. This observation is contrary to the experience of the forensic mtDNA community. There are several explanations for the unusual findings of high levels of heteroplasmy. First, the template quantities of DNA are approximately three orders of magnitude higher than required for mtDNA sequencing, and an excessive number of amplification cycles were used. Thus, the protocol used did not follow routine practices by the forensic community. Second, there are misidentifications and tabular errors that call into question the reliability of the findings. Third, by comparing the natural human mtDNA variation with a reference sample population with that observed in the heteroplasmy in hair study, the data are inconsistent with population genetic expectations. The observation of high levels of heteroplasmy may be due to contamination of the samples and/or possibly the amplification of nuclear pseudogenes. The results observed in the heteroplasmy in hair study do not apply to other methods of mtDNA analysis and cannot be used to question the reliability of the current forensic mtDNA practices.     

单管一步甲基化可变位点分析技术

目的建立单管一步甲基化可变位点（methylationvariableposition,MVP）分析技术一单管消化后PCR链融解曲线分析（post—digestionPCR—meltingcurveanalysis,PDP—MCA）。方法以文献报道的差异甲基化区（differentiallymethylatedregion,DMR）为模型,在MVP两侧设计一组解链温度各不相同的引物。应用FastDigest甲基化敏感性限制酶（methylation—sensitiverestrictionenzyme,MSRE）,在同一反应管内顺次进行DNA的酶切、复合扩增和MCA检测．生成MCA图谱。同时用该方法和传统的MSRE—PCRMCA技术检测相同样品（外周静脉血、精液、阴道液各5份）,比较两种方法检测结果,验证其可行性,并分析比较不同样品的MCA／HRM图谱。结果解链温度相差2℃以上的片段,MCA峰分离良好,复合扩增后可以用MCA技术检测。应用单管PDP—MCA技术,可以集酶切、扩增和检测三步于一管,在2h内得到与传统方法一致的特异性图谱和数据,并实现样品的快速分类鉴别。结论单管PDP-MCA技术可以实现多个MVP的单管、闭管检测,具有简便、快速、易于自动化等优点,可用于样品DNA甲基化差异的检测。     

mtDNA—HVⅠ和细胞色素b片段的复合扩增及其法医学应用

目的探讨复合扩增mtDNA D环HV I和细胞色素b片段进行种属鉴定和个体识别的方法及mtDNA-HV I多态性。方法用两对引物同步扩增HV I片段与细胞色素b片段,银染显带检测扩增产物,ABI377测序仪及荧光测序技术分析扩增产物序列多态性。结果人类有279bp,358bp两条带,动物只有358bp一条带。通过对131例随机广东汉族人群个体进行mtDNA控制区(15997～16236))序列测定统计,得出此区域的序列多态性。共发现69个位点变异,平均每个个体存在2.679个碱基突变,检出67个单倍型,基因多样性为97.92％。结论mtDNA控制区(15997—16236)具有较高的序列多态性。为良好的个体识别标记。复合扩增mtDNA D环HV I与细胞色素b片段进行测序分析可以同步进行种属鉴定和个体识别。