| Crime scene samples such as blood stain, seminal stain, saliva stain, hair and so on are tested and analyzed by Deoxyribonucleic acid (DNA) inspection technology to define and exclude suspects and in the practice of the public security case plays a pivotal role. The test process usually includes the treatment of biological material evidence, DNA extraction, multiplex polymerase chain reaction (PCR), and capillary electrophoresis for short tandem repeats (STR) which took roughly 10 to 12 hours. It could not only reduce inspection personnel work but also further improve the inspection speed to meet the timeliness requirement of case investigation and litigation if the process times could be shorted and the operation steps could be simplified. Biological material evidence after pretreatment for DNA need multiplex PCR amplification of the STR systems to get autosomal or Y chromosome STR profiling, this is the conventional method of forensic DNA testing. PCR is one of the most commonly used molecular biology techniques which were used through the cycle of degeneration, annealing and extending to amplify the nucleic acid molecules. Ordinary PCR amplification process generally takes about 3 hours, which is one important limit in the inspection process of biological material evidence. It will significantly improve the speed of material evidence examination if we can shorten the PCR process time. Rapid PCR is one process to amplify the nucleic acid molecules in a shorter time which is based on the principle of ordinary PCR under the premise of ensuring reaction specificity, sensitivity and fidelity. Recently, the rapid PCR were investigated respectively from three aspects:polymerase improvement, additives research and innovation creation for the thermal cycler. Rapid amplification DNA polymerase is one high temperature DNA polymerase of high extensibility and rate which is obtained by genetic engineering. Now SpeedSTARTM HS of TAKARA company, PyroStart Fast PCR Master Mix of Fermentas company, KAPA2G Fast PCR Kits of Kappa company, etc were the commercial rapid polymerase commonly. Different polymerases show differences in fidelity, specificity, etc. Through the rapid heat transfer design patterns or increase the rate of heat conduction, the 1/cooling rate of the instrument could increased to 6-15 ℃/s. Such as the SpeedCycler2 of Germany Analytik Jena company, Mastercycler pro of Eppendorf company, etc. Rapid amplification technique has been used in pathogen detection, diagnosis, and other fields, but there is few studies in the applications of forensic science. To ensure the specificity accuracy, sensitivity, and other important parameters are not affected is the first thing in the study of rapid PCR technology system establishment, and then improve the amplification rate.Currently, trace touch biological material evidence are as problems in case and hotspots and difficulties in the field of forensic genetics research. Skin or mucosa cells can be left on items of the crime scene which are called trace touch biological material evidence. Skin exfoliation cells or epithelial cells can be left on many items such as knife handle, key, phones, clothes, hats, shoes, socks, gloves, door handles, kernel, toothbrushes, beverage can and so on. These potential biological material evidences may be the key in case. But it is difficult to retrieve usable profiles from such samples and repeated experiments are needed, which become the limiting step in case test. Along with the improvement of criminal intelligence, obvious biological material evidence such as blood stain, hair, seminal stain etc. are often consciously destroy, but trace touch biological material evidence because of the little quantity which is not easily identified tend to be ignored. Such samples get more and more attention and the number of inspection also have been grown since the first report of touch DNA by van Oorschot etc. in 1997. For example, more than forty thousand DNA materials need inspection each year in the past two years of which about 40% for the trace touch biological material evidence in the Institute of Forensic Sciences, Ministry of Public Security of China. It is difficult to see the location of cells or DNA, such evidence usually be processed blindly and target cells of samples are not obtained sometimes. If too much samples are cut, DNA concentration could be decreased, sometimes contamination problem may arise, so the detection rate is very low. Although the collection and extraction device of biological exfoliated cells have been developed during the period of "11th five-year plan" in our country, the systematic fundamental theoretical research for such evidence is still lack of. In fact, big differences were existed in DNA quality deposited on the surfaces of different substrates such as porous and non-porous substrates, storage time, the incidence of secondary transfer, application of DNA preprocess method and so on. Systematic research and the accumulation of basic data of related issues can effectively avoid blind and repeated inspection, and has important guiding significance in rapid disposal and inspection strategy such as test sequence, the parts DNA extracted, preprocess methods and so on. As we know, crime scene samples after their collection are packaged in bags which were specified by public security agencies and transported to the laboratory for examination and DNA analysis. Locard’s exchange principle suggests that every step, every contact, every touch leaves an unavoidable, unbiased, irrefutable evidence that will "serve as a silent witness", only diminished by human failure to understand and wholly utilize its value. Significantly, the DNA transfer will happened when the biological material contacting with the evidence bags in the process of packaging and transportation which has been called secondary transfer, and the biological material collected from a crime scene is usually the result of primary transfer and in these process, secondary, tertiary and further transfer may also happen. In recent years, the DNA source of trace touch biological material evidence has been an important research:from the cell-free DNA or intracellular DNA.On early basis, we use hand touch samples as research object for trace touch biological material evidence to study the DNA detection and inspection results, the best preprocess method and the change of detection and inspection effect after storage at different times. To investigate whether DNA secondary transfer happened within packaging in different bags, the differences in transfer rates, the existence of cell-free DNA, and the proportion of cell-free DNA vs. intracellular DNA. According to different rapid polymerase and different thermal cycler instrument, we select adaptive polymerase and established rapid amplification system which is commonly used in forensic science based on the primers of AmpFLSTR Identifiler Plus kit and primers with 19 loci made by Institute of Forensic Sciences, Ministry of Public Security of China. To establish rapid testing technology for such samples through systematic exploration of basic theory problems on DNA content, storage time, source, secondary transfer and related issues on rapid PCR, two parts were investigated in this study:the rapid disposal part and the rapid amplification part.For the study on trace touch biological material evidence:DNA was extracted from mock exhibits after using four preprocessing methods (directly cutting method, stubbing procedure, vacuum cleaner method and double swab technique) and the best preprocess protocol was used to deal with the exhibits after storage for different lengths of time (2,6,10,30,60,90,180,360 days) and to observe the differences on the detection and inspection results as time goes by. For the secondary transfer study, this investigation mock exhibits and simulates the transportation process of biological material evidence using the seal-lock plastic bags and yellow paper envelopes. DNA extracted from the samples and material evidence bags were quantified and amplified using standard manufacture’s procedures and the results were analyzed and discussed to observe the differences of transfer rates among different biological material evidence and bags. For the cell-free DNA study, we mock hand, face, foot trace touch exhibits and mouth swabs. The samples were dip in water and both intracellular DNA and cell-free DNA were quantified and amplified using standard manufacture’s procedures, the results were analyzed and discussed to observe the existence of cell-free DNA, the proportion of cell-free DNA vs. intracellular DNA and how the cell-free DNA affects the final DNA typing。 The ambient temperature in the laboratory was maintained between 22 and 24℃, with a mean relative humidity of 50% during the experiments. DNA was processed using procedures identical to those used for case work samples. Briefly, DNA was extracted using a Mag AttractTM 48 DNA Manual kit (Qiagen), quantified using a QuantifierTM Human DNA quantification kit (Applied Biosystems) and 7500 Real Time PCR system (Applied Biosystems), and genotyped using an AmpFLSTR Identifiler Plus kit (Applied Biosystems), ABI 3130XL Genetic Analyser (Applied Biosystems), and Gene MapperTM ID V3.3 Software (Applied Biosystems) using the manufacturers’ protocols. Both positive (9947) and negative controls were implemented for all reactions during the DNA processing steps. The results are presented as the DNA quality rather than the DNA concentration. A calling threshold of 50 RFUs was used for analysis. The calculation of average alleles was the alleles detected divide the number of samples and the detection rate was the number of samples detected divide the number of all samples. The Student’s t test, One-Way ANOVA, Mann-Whitney U-test and Kruskal-Wallis one-way analysis of variance were used to analyze the data. Finally, We found that the amount of DNA by using the direct cutting method was greater than that by using the double swab technique and the vacuum cleaner method. Four preprocess protocols show differences among different substrates. As time goes on, the amounts of DNA after placed various days have been no significant differences in 360 days. In the secondary transfer study, we found that the average transfer rate of the amounts of DNA detected was 22.06% and 100% of the exhibits were transferred. For the seal-lock plastic bags, the average transfer rate was 17.85%, and 26.27% for the yellow paper envelopes. Significant difference of the amounts of detected DNA between the seal-lock plastic bags and yellow paper envelopes was observed (p= 0.023). The transfer rates of the yellow envelopes were higher than that of the seal-lock plastic bags. Significant difference of the transfer rates between the trace touch biological material evidence and other samples was observed (p= 3.14E-19), the transfer rates of the touch evidence were higher than that of other samples. In the cell-free DNA study, the cell-free DNA was detected on 75.6%of the samples. The detection rates in hand, face and foot touch samples, and mouth swabs were 82.1,82.1,53.6, and 92.4%, respectively. The amount of cell-free DNA was 12.9% of the amount of total DNA in trace touch samples.For the study of related issues on rapid PCR:Two thermal cyclers, seven rapid polymerases and two mixed primers of commonly used STR amplification kits were selected to perform this study. To optimize the enzyme of shortest amplification time and best amplification effect by adjusting, comparing and optimizing the reagent, primers and two thermal cyclers to reduce amplification time and improve amplification effect. A series of validation and evaluation were performed for the optimized amplification system. This study established a 10μL amplification system on SpeedSTAR enzyme of TAKARA company and primers of AmpFLSTR Identifiler Plus kit which took only 25 min on a thermal cycler. Meanwhile, a 10 μL amplification system of 69 min was established on FastStart enzyme of Roche company and mixed primers of 19 loci with a thermal cycler. Two systems can be applied on routine samples such as blood, mouth-swabs, exfoliated cells, seminal stain, bones, teeth etc., the sensitivity and accuracy were 0.25 ng and 100%.Conclusions:The study on trace touch biological evidence showed that the detection and inspection results of DNA by using different preprocess protocols show differences on different substrates. The amounts of DNA on porous substrates were higher than that of non-porous substrates. Four preprocess methods showed differences on the seven substrates and under normal and dry conditions, the amounts of DNA and inspection results of trace touch biological material evidence showed no significant trends as time goes on in 360 days. DNA transfer and potential loss occurred within forensic exhibit packaging and transportation. Differences of the transfer rates were observed among different samples and bags, the transfer rates of the yellow paper envelopes were higher than that of the seal-lock plastic bags and these transfers affect the final DNA typing. The cell-free DNA did exist extracellular, and the amount of the cell free DNA occupy a proportion in the trace touch biological material evidence. Trace touch biological material evidence occupied main parts in case in recent years, and this study will help to guide the inspection process and preprocess methods dealing with such samples to provide a new approach and data for rapid test and inspection necessity of samples after storage at different times, provide a reliable theoretical basis and data support for the public security institutions and research institutes, improve the probative force of physical evidence and strong support for case broke and scientific research. Even little loss of DNA may lead to part or no DNA profiling which may be the key to the case especially for now, trace touch biological material evidence have been one of the most common evidence. Dry samples were used in this study and moisture influence was ruled out which could provide new insights and help to the extraction, storage and transportation of dry samples. Along with further study of the influences on different substrates, wet and dry samples and different conditions, it would provide more convenient and suitable method for storage, extraction and transportation on samples. Research on cell-free DNA of trace touch biological material evidence investigated the existence of cell-free DNA, the proportion of cell-free DNA and intracellular DNA and evaluated the profiling results of cell-free DNA. The study found that the quantity and profiling of cell-free DNA is lower than that of the intracellular DNA which provide important arguments and evidence on such samples and scientific basis for related technology and device research though cell-free DNA were a bit poor in case. Rapid PCR experiments were performed with usual kits and seven rapid enzymes on two thermal cyclers by shortening the amplification time and adjusting the amplification system which systematic investigated and optimized the seven rapid enzymes and mixed primers of 19 loci. Two optimized amplification systems were established which have advantages in shortening the PCR amplification time and shorten the time of total inspection process of DNA. This study improve the efficiency of forensic science, meet the requirement of case investigation and litigation for timeliness, enhance the value of biological material evidence, provide convenience for PCR amplification for the public security organ and meet the requirements of scientific research institutes for PCR amplification. Through systematic research, selected, establish and optimize the amplification system which is suitable for all kinds of routine forensic material evidences, a foundation was laying for further research and improvement on rapid enzyme and thermal cycler. Overall, this study systematic investigated the rapid disposal strategy of trace touch biological material evidence, and the experimental result finally formed rapid disposal strategy which was suitable for such samples, so as to improve the efficiency of forensic examination and the probative value of biological material evidence. |
