Saliva and saliva spot inspection

钟树荣（ PhD ，副教授）昆明医科大学法医学院

Cell Phone ： 18213823198 E-mail ：

zhongshurong@hotmail.com

What are the objects found saliva stain easily ？

bite mark

一、 The characteristics of the saliva and saliva stain

• （一）、 saliva

• (saliva) is secretion of the salivary glands, saliva is colorless, tasteless, and is close to neutral liquids.

saliva

无机物质

有机物质

water （ 99.4 ％）

Sodium, potassium, chloride, calcium

Phosphoric acid

Carbonic acid

rhodanic acid

Blood type thing

Mucin, ball eggs

Amino acids, urea, urine

淀粉酶麦芽糖酶

• Fall off in addition, the saliva and oral mucosa epithelial cells, a large number of bacteria, a small amount of white blood cells, and food scraps, etc

• Saliva stain is saliva drying after the formation of the spot mark, saliva spot is more common in forensic inspection of one of the biological material inspection

• Saliva spot on a white background with pale yellow, Pale blue fluorescence under ultraviolet light

（二）、 Saliva spot

Confirm the saliva spotConfirm the saliva spot

亲权鉴定亲权鉴定个人识别个人识别（（ Individual IdentificationIndividual Identification ）） （（ parentage testingparentage testing ））

二、 Saliva and saliva spot inspection purpose

Confirm whether or not

Saliva spot

Salivary amylase Check

out the test

三、 Saliva stain proof

Fall out of the oral mucosa

epithelial cells Check out the test

starch Flecked leaching liquid

iodine

The same blue Change the blue

In the spot amylase In the spot amylase

Prove the existence of sugar

theory

（一）、 Saliva amylase detection test

Starch iodine turned blue

碱性铜氯化三苯基四氮唑

Flecked unchanged leaching liquid reaction with starch and iodine blue, can only prove flecked contains amylase, doesn't mean the blackspot is saliva.

matters need attention

（二）、 Fall off oral mucosal epithelial cells of the inspection

cell membrane

nucleus

Cytoplasm

Flat, polygons, the shape is not very regular

Cell membranes are thin, coloring shallow in the cell membrane is the cytoplasm, dyed deep shape larger is the nucleus.

HEDyeing

Acid fuchsin methylene blue staining

attention：

Because saliva spot check material often appear in People's Daily supplies Or items, such as cups, cigarette butts, stone, etc., so confirmed Saliva after the spot, can be directly used to identify the individual, don't have to Species identification.

But when determining the bite of saliva spot, first of all want to make sure Is a person or animal bites.

Determination of saliva secretion spot ABO blood group and the state

Polymorphisms of proteins and enzymes in saliva spot detection

Sex identification of saliva

Saliva of DNA polymorphism analysis (individual identification, paternity test)

四、 Saliva of personal identification

Carrier Type DNArelative amount

About the data

Fresh saliva many 0.8ug/ml

Sterile physiological salt Water gargle water

more 1.5ug/ml

Saliva swabs few 0.26ug/cm2

Saliva spot less 0.16ug/cm2

cigarette end less 12-120ng/ 个

The PCR technology can enlarge the existing DNA is extremely limited to 2 ng, Reason for common saliva (spot) for examination for DNA testing is absolutely Practical and feasible.

The extraction of DNA specimens

1. Biological samples and the

separation of biological

components (carrier)

2. DNA extracted from cells

and cells with other

components

概述

How to obtain nuclear DNA ?

summarize

Nuclear DNA extraction principle

Raw material cells

SDS Heated to boil

nucleus

hypotension

Nuclear membrane rupture, released from within the nuclear DNA

Proteinase

Hydrolysis of DNA binding proteins, DNA free in solution

SDS

Phenol and chloroform, ethanol

Magnetic beads, silica gel

Impurity removal, collecting DNA

概述

（ 1 ） Maximum remove the RNA in the sample DNA

solution, the composition such as proteins,

polysaccharides and lipids molecules;

（ 2） As far as possible keep the integrity of the extracted

DNA molecules;

（ 3) to reduce or eliminate the possible of enzyme

inhibition substances, such as organic solvents,

detergents, metal ions, and dyes, and other enzyme

activity by inhibiting DNA analysis.

The ideal DNA extraction

summarize

五、 Saliva and saliva of extraction

（ 1）、 Fresh saliva collection ：

First gargle, gauze or cotton swabs to be included in the oral cavity, take out after soaking, dry in ventilation places immediately cool, package equipment inspection, respectively.

If gathering flow of saliva, because it contains blood enzymes, shall be immediately put in boiling water bath (bath boil 10 min) inactivated enzyme type material.

（ 2） . Saliva (spot) extract on the site

Found at the scene of the cigarette butts, paper towels, cups, etc should be wearing gloves or with tweezers collection, packaging, respectively.

The scene suspected phlegm article such as stains, skin and bite marks, soaked with saline water gauze wipe patches, transferred to dry patches on the gauze shade, mark

respectively for inspection.

Second step transfer method

• Key to the success of saliva spot transfer ：

Drink or jelly contact people in the sales

process, the scene of the mug is not only the criminals used, large area extraction line won't work for sure. This requires inspection case personnel should through the simulation experiment in extraction for examination or normal life habit that target area.

Several kinds of common carrier transfer of saliva

on the spot

With a handle mug

Cup without handle

（ 1 ） pouring cup

（ 2 ） Mineral water bottle

（ 3 ） butt

（ 4 ） Jelly shell

0.3 － 0.5cm

（ 5 ） bite mark

Generally associated with traces of saliva when occlusion, so it cannot be ignored Wound saliva spot inspection. Found after a bite, use dry first Net gauze to cover, to prevent pollution. Then wipe with two step method Pass the effective components in saliva stain. Do not use hand before inspection Wipe, also don't blot out drugs.

（ 6 ） Sunflower seeds

Due to the different people's habits, into hard seeds with shell on the adhesion of the mouth How much cavity epithelium also will be different. But according to the habits of most people , is to use teeth clacking, so it contains relatively few components . Sunflower seeds in the fiber and has strong adsorption, should not be so Should use the method of transfer cells, direct access to the sunflower seeds. In addition, Sunflower seeds on the surface due to contain pigments, add baking in the process of making And have serious effects on amplification so using chelex extracted to The DNA to do further purification and enrichment.

（ 7 ） Gum or coffee candy

Summary

Saliva and saliva spot inspection:

• perusal

• Saliva stain proof (amylase, shedding of the oral mucosa epithelial cells)

• DNA polymorphism analysis (individual identification and paternity tests)

• draw