Alpha-amylase kinetic test in bodily single and mixed stains

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

Review: The physiology of saliva and transfer of drugs into saliva

Although saliva or oral fluid “lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears”, quoting Mandel in 1990 [I.D. Mandel, The diagnostic uses of saliva, J. Oral Pathol. Med. 19 (1990) 119–125], it is now meeting the demand for inexpensive, non-invasive and easy-to-use diagnostic aids for oral and systemic diseases, drug monitoring and detection of illicit use of drugs of abuse, including alcohol. As the salivary secretion is a reflex response controlled by both parasympathetic and sympathetic secretomotor nerves, it can be influenced by several stimuli. Moreover, patients taking medication which influences either the central nervous system or the peripheral nervous system, or medication which mimic the latter as a side effect, will have an altered salivary composition and salivary volume. Patients suffering from certain systemic diseases may present the same salivary alterations. The circadian rhythm determines both the volume of saliva that will and can be secreted and the salivary electrolyte concentrations. Dietary influences and the patient's age also have an impact on composition and volume of saliva. The latter implies a wide variation in composition both inter- and intra-individually. Sampling must therefore be performed under standardized conditions. The greatest advantage, when compared to blood sample collection, is that saliva is readily accessible and collectible. Consequently, it can be used in clinically difficult situations, such as in children, handicapped and anxious patients, where blood sampling could be a difficult act to perform.     

microRNA Detection in Blood,Urine, Semen,and Saliva Stains After Compromising Treatments

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.     

A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice.     

唾液中乙醇含量检测试剂条的研究

目的根据唾液和血液中乙醇含量相关性的实验结果,建立了一种简便快速、准确可靠的检测唾液中乙醇含量的方法。方法本方法利用酶学原理,将一定量乙醇氧化酶(ALO)和过氧化物酶以及底物四甲基联苯胺(TMB)固定于试剂条上,当样本中含有乙醇时,酶学反应使底物TMB显色,通过比对反应的不同颜色,对样本中乙醇质量浓度进行半定量。结果用本方法检测300个自愿者的唾液,和用GC/MS法对照检测志愿者唾液中的乙醇含量,定量结果基本一致。本产品检测过程仅需2min,其检测的阈值为0.1mg/mL,敏感度为96.5%,特异性为91%,准确性为94.7%。结论采用酶学方法制备的乙醇含量检测试剂条,通过显色反应对唾液中的乙醇含量进行半定量检测,其特点为快速简便、准确可靠,适合现场使用。     

The Differentiation of Menstrual from Venous Blood and Other Body Fluids on Various Substrates Using ATR FT‐IR Spectroscopy

Crime scene investigators and laboratory analysts use chemical tests to detect and differentiate body fluids. Testing often requires a sample of the stain, and the chemicals may cause degradation of the fluid or interfere with subsequent tests. Colorimetric chemical tests do not differentiate between different types of the same fluid, such as venous and menstrual blood, and there is no presumptive test available to simultaneously differentiate several body fluids. In this study, we recorded ATR FT ‐IR spectra of venous and menstrual blood, semen, saliva, and breastmilk. Neat and simulated casework body fluid samples were analyzed on cotton, nylon, wood, paper, and glass substrates. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences. Venous and menstrual blood is differentiated by the peak at 1039 cm^?1 attributed to phosphoric acid found in menstrual blood. Peak intensity is influenced by the porosity and weave of the substrate fabric.     

A Rapid,Confirmatory Test for Body Fluid Identification,

We have developed a technique that allows investigators to confirm the presence of blood, semen, and/or saliva in a crime scene sample. It is a confirmatory test where multiple samples can be processed in less than an hour, and it is potentially portable, permitting samples to be processed at the crime scene. Samples at a scene giving a positive result can be further processed while those failing to do so may be ignored. There is a large and growing backlog of DNA evidence in the USA, slowing down the criminal justice system. This backlog has continued to grow despite an increase in the ability to process evidence faster. This technique uses quantum dot molecular beacons to test for tissue‐specific RNA species, identifying particular body fluids. We have demonstrated the tissue specificity of molecular beacons for blood, semen, and saliva.     

唾液检材在毒品检测中的应用

近年来,毒品滥用问题日益突出,提高生物样品中毒品检测技术的性能是法庭毒物学研究的重点。相比于血液和尿液样品,唾液在的样品采集和毒品检测中具有诸多优势,因而逐渐受到重视。本文对近年来国内外唾液样本用于毒品检测方面的研究成果进行综述,介绍唾液毒品检测的发展情况以及相关的代谢动力学研究状况,旨在为相关研究和实践提供参考。     

唾液中滥用药物分析及其与血液中药物浓度的相关性

唾液是一种成分简单、易于采集的体液,某些药物在唾液中的浓度可以反映其血药浓度。本文分析了滥用药物进入唾液的机制和影响因素,综述了唾液中滥用药物分析时样品的采集、前处理和检测方法以及唾液与血液中药物浓度的相关性。认为唾液是临床和法医学方面很有价值的分析样品,用唾液中滥用药物浓度来推测血药浓度具有一定的法医学意义。