Diatom UPA (universal plastid amplicon) gene analyzing method and application thereof in legal medical expert detection
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A genetic analysis, diatom technology, used in the field of forensics
Inactive Publication Date: 2017-09-26
GUANGZHOU CRIMINAL SCI & TECH RES INST
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There is still controversy in the academic circles about how to judge whether the corpse in the water entered the water before death or was thrown into the water after death
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Embodiment 1
[0025] Example 1 Primer Design and Proof of Primer Specificity
[0026] A pair of upstream and downstream primers were designed according to the UPA gene of diatoms:
[0027] Upstream primer (as shown in SEQ ID NO.1):
[0028] 5'-CAGTGAGATACCACCCTTGTAATGTT-3'
[0029] Downstream primer (shown as SEQ ID NO.2):
[0030] 5'-TCTAGATACGATTTCTAACCGCTCAGA-3'
Embodiment 2
[0031] Example 2 Specific verification of primer pairs
[0032] Use the above primers and probes to perform real-time fluorescent quantitative PCR detection of DNA from different samples. The 5' end of the probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a NFQ-MGB fluorescent quencher group. Five common silicon Algae, two human commensal bacteria and a variety of non-diatom algae DNA for primer specificity verification.
[0034] obtained after amplification figure 2 Amplification curves are shown. attached by figure 2 It can be seen that...
Embodiment 3
[0036] Embodiment 3 Animal group control experiment
[0037] Samples of lung, liver and kidney (organs of the systemic circulation) of the artificially drowned experimental pig group (n=15), the experimental pig mechanically sacrificed into the water group (n=15) and the blank control group (n=5) of non-drowning experimental pigs Under the same amplification system, the genomic DNA extracted from the sample was amplified by real-time fluorescent quantitative PCR with the primer probe designed in Example 1. It was stipulated that an amplification curve appeared and the Ct value (Cyclethreshold) was less than 35, which meant positive amplification, and the result was judged as The detection and detection rate results are shown in Table 1.
[0038] Table 1 The number of cases and positive rate of diatom UPA gene detected in the organs of animal circulation in each group
[0039]
[0040]As can be seen from the above experimental results, using the method of the present invent...
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Abstract
The invention discloses application of a diatom UPA (universal plastid amplicon) gene in judging whether the drowned dead really die of drowning. The invention provides a pair of primer pairs for detecting the diatom UPA gene and further provides a diatom UPA gene analyzing method. The analyzing method comprises the steps of extracting diatom nucleic acid, utilizing the primer pair shown in the right of claim 2 to amplify the UPA gene in the nucleic acid, utilizing an amplified product to prepare a sample to be tested and performing nucleic acid analysis on the sample to be tested. Furthermore, a used probe is 5''-CGGAGGCGTACAAAG-3'', the 5'' end of the probe is marked by an FAM fluorescent reporter group, and the 3'' end of the probe is marked by an NFQ-MGC MGB fluorescent quencher group. The invention further provides application of the diatom UPA gene analyzing method in legal medical expert detection. The application of the diatom UPA gene analyzing method in legal medical expert detection comprises the steps of extracting the diatom nucleic acid in organs of the drowned dead, amplifying the diatom nucleic acid through a PCR technology, then detecting and comparing the diatom nucleic acid with diatom nucleic acid in a water sample. Through comparison of relevance ratio and a sequencing result of the diatom UPA gene, whether the drowned dead really die of the drowning can be judged. Meanwhile, the application of the diatom UPA gene analyzing method in legal medical expert detection is beneficial to finding out a real drowned area.
Description
technical field [0001] The invention relates to the field of forensic science, in particular to an analysis method of diatom UPA gene and its application in forensic detection. Background technique [0002] In the practice of forensic investigation, we often encounter the inspection and identification of drowning-related cases. Due to the many rivers, lakes and seas in our country, the complex environment, and the influence of factors such as corpse corruption, the identification of the cause of death of corpses in water has always been one of the difficulties in forensic practice. There is still controversy in the academic circles about how to judge whether the corpse in the water entered the water before death or was thrown into the water after death. Diatom testing has been the most commonly used method in forensic practice. At present, there are many diatom detection methods, but the commonly used diatom detection methods in China are mainly based on diatom morphology....
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