唾液酯酶多态性在个体识别中的应用研究

目的 探讨唾液酯酶（ Set）多态性在法医学亲权鉴定和个体识别方面的应用价值。方法 应用聚丙烯酰胺凝胶盘状电泳及固蓝 RR染色方法,调查了 114名中国人 Set的表型分布及基因频率,用χ 2检验进行统计学分析。结果 中国人酯酶表型频率 Set F 22.81％, Set FS 50.88％, Set S 26.31％ ;基因频率为 SetF 0.482 5, SetS 0.517 5;非父排除机率为 0.187 5,个体识别率为 0.619 9。结论 Set有较高的父权排除率和个体识别率,可作为法医学亲权鉴定和个体识别的重要标记系统之一。     

Determination of phenotypes of salivary amylase in liquid saliva and saliva stains

An isoelectric focusing method is described for typing salivary amylase in liquid saliva and saliva stains. The estimated gene frequencies in a British population, calculated on the basis of three alleles operating at a single locus, were Amy 1, 0.909; Amy 2, 0.065; Amy 3, 0.026. This system may be useful in forensic investigations.     

A novel method for the identification of saliva by detecting oral streptococci using PCR

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.     

人唾液中苯丙胺类药物检测方法进展

目前人唾液中苯丙胺类药物的检测研究较少,鉴于唾液检材具备很多的实用价值和优势,本文综述了国内外对唾液检材中苯丙胺类药物的检测研究进展,重点包括免疫分析筛选方法、液-液萃取法、固相微萃取法等萃取方法以及气相色谱/质谱联用法、液相色谱/质谱联用法等确证方法的发展情况。     

A sensitive and simple assay of saliva on stamps

Identification of saliva on stamps or envelope flaps remains yet a not widely studied problem. In most forensic laboratories it is seldom carried out, but this fact does not reduce the importance of the assay. Most authors consider amylase a sufficiently specific marker of the presence of saliva; really, the only other human body fluid that contains high amounts of this enzyme is the pancreatic juice (and therefore feces). Here we present a simple and sensitive assay for the determination of alpha-amylase that uses a commercially available and well-known substrate. It is hydrolyzed by amylase with the production of soluble blue fragments, that can be measured by photometry, obtaining objective results. The presented assay identifies 1 X 10(-6) diluted saliva or that present on 0.5 mg of a stamp; 16-year-old samples can also be identified. Intra-assay and day-to-day CV resulted in 10.8% and 13.7%, respectively. Owing to the high sensitivity of the test, handling samples or reagents can introduce contamination with saliva traces, giving false-positive results. Addition of EDTA 0.1 mol/l to the incubation mixture, lowering the sensitivity to 1 X 10(-3) diluted saliva, overcomes this problem.     

Detection of ABH blood group antigens in the saliva of Koreans and their stability according to storage of saliva samples

The purpose of the present study was to identify salivary molecules carrying the ABH blood group antigens in Koreans and to investigate the changes in these antigens according to processing and storage of saliva samples. Secretor or non-secretor phenotypes and salivary components carrying the ABH antigens were identified in 90 subjects, 30 subjects in each ABO blood group, by SDS-PAGE and immunoblotting. Saliva samples were then obtained from 12 secretors-two males and two females in each ABO blood group and aliquots of both fresh saliva samples and their supernatants after centrifugation were stored at room temperature, 4, -20 and -70 degrees C. The same experiments were performed after 1, 3 and 6 months to investigate changes in the blood group antigens. In all 68 secretors, high-molecular-weight salivary mucin (MG1) was found to be the primary carrier of the ABH antigens. A salivary component of approximately 80 kDa also carried H antigen in seven saliva samples of 22 blood type O secretors. The blood group antigens were better detected in centrifuged samples. In saliva samples preserved at room temperature and 4 degrees C, the blood group antigens were either not detected or detected as degraded molecules. No change was found in the blood group antigens in saliva samples preserved at -20 and -70 degrees C for 6 months.     

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.     

The sensitivity and specificity of red-starch paper for the detection of saliva.

The detection of saliva in forensic casework is extremely important in many types of cases. This study describes a relatively sensitive method, based on a red dye bound to starch, for the detection of amylase. The sensitivity and specificity of the method has been examined by testing over 50 household products, various body fluids and five laboratory chemicals. This study demonstrated for the first time that positive results can be obtained from certain washing powders as well as other household products. As well as detecting amylase in saliva, positive Red-Starch results were obtained from faeces and urine. The method was found to be suitable for the detection of mixtures of saliva and semen in conjunction with the Brentamine test for the detection of acid phosphatase.     

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。     

Detection of delta 9-THC in saliva by capillary GC/ECD after marihuana smoking

A method is described for the determination of delta 9-tetrahydrocannabinol (delta 9-THC) in the saliva by the use of a combination of moving-precolumn injector and glass capillary gas chromatograph with electron capture detector (GC/ECD). There were no interfering peaks due to impurities around the peak of pentafluoropropyl derivative of delta 9-THC (delta 9-THC-PFP). This GC/ECD method was linear over the range of 5-200 ng/ml of delta 9-THC-PFP. The lower detection limit was approximately 1 ng/ml. delta 9-THC content in the saliva after experimental marihuana smoking was measured by this method. It was demonstrated that for at least 4 h after smoking the level of delta 9-THC was sufficient for detection.     

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.     

Immunofixation of complement component C3 phenotypes in bloodstains after cellulose acetate electrophoresis

The determination of the polymorphic C3 phenotypes was accomplished by electrophoresis on cellulose acetate followed by immunofixation. The three common phenotypes resulting from the two common codominant alleles C3S and C3F were clearly distinguishable in blood and bloodstain samples. Storage and degradation of C3 in blood samples as well as the stability of C3 in dried bloodstains is discussed.     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

Amylase levels in semen and saliva stains

Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.     

The detection of gamma globulin and Inv-factors in semen and saliva and of haptoglobins in semen (author's transl)]

Gm- 1-, 2-, and Inv 1-factors can be demonstrated in semen and saliva. For the examination of traces it is necessary to verify the suitable dilutions for the different charges of antisera and to test the eluates of the samples if they have a sufficient concentration. We observed some incorrect negative results in seminal and saliva stains which were apparently caused by insufficient material. The demonstration was independent of the secretor type. Haptoglobin could not be determined in semen and saliva.     

Detection of saliva traces using test strips

The test strip Rapignost-Amylase (Behring) for the rapid determination of alpha-amylase in the urine is also suitable for the determination of salivary amylase in stains stored up to 6 weeks at room temperature. The stains are extracted with physiological saline (extraction time 30 min), then the application zone of the strip is wetted with the extract. Positive amylase-reaction is recognised as a reddish-violet colouration of the reaction zone. Biological stains with low amylase concentrations (urine semen, vaginal secretion, mucus) react amylase negative. The method is uncomplicated and can be completed within 30 min. The test strips are easily available and stable during storage. Therefore the determination of saliva with test strips should be preferred to the clinical methods if the storage times of the stain are not longer than 4-6 weeks. It is a suitable procedure to determine salivary stains for use in forensic biology.     

Radioimmunoassay detection limits for 19-OH F1 alpha/F2 alpha prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F1 alpha/F2 alpha (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 microliter of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 microliter semen. The assay could be conducted over a pH range of 7.5-10.5 even after the PG has been heated to 100 degrees C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 microliters of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 microliters of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.     

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.     

Addressing the alternate hypothesis: Transfer and persistence of saliva beneath fingernails

This study investigated the transfer and persistence of salivary DNA under fingernails. This was performed to address a common alternate hypothesis presented to scientists in court, asserting that a relatively large quantity of DNA detected beneath the fingernails, typically from a victim of crime, originates from innocuous transfer of saliva in a casual setting.It was determined through these studies that contact with liquid saliva was an effective way to transfer foreign DNA beneath fingernails. However, when saliva was dried, DNA did not readily transfer through casual contact.When liquid saliva was placed directly beneath fingernails the amount of DNA detected from the saliva donor twenty-four hours later was several hundred-fold lower than the amount detected when sampling occurred immediately following deposition. Furthermore, when the recipients’ hands were washed immediately following the deposition of liquid saliva beneath fingernails, the majority of foreign DNA was removed following one hand washing and all detectable foreign DNA was removed from most recipients’ hands after three or six hand washings.This study demonstrates that casual contact with wet saliva can result in the transfer of substantial quantities of DNA beneath fingernails but that it does not typically persist for extended periods of time and is mostly removed if the hands are washed soon after deposition.