当代法医科学取决于DNA分析，将个人与犯罪现场联系起来。DNA的来源包括包括唾液在内的体液。淀粉酶是人类唾液中的主要酶，因此，如果检测到，则表明可能存在人类唾液。淀粉酶纸可用于绘制明显的唾液，因此提供了可以从中提取和分析DNA的来源。在这项研究中，首先，使用淀粉酶标准的稀释液测试了淀粉酶纸的敏感性，并随后还使用新鲜的人类唾液进行了测试。总共进行了三项试验，使用淀粉酶标准的前两个使用新鲜唾液。前两个试验首先证明了淀粉酶的检测取决于沉积淀粉酶的材料。第三次试验表明，人类唾液中的淀粉酶水平可能会显着下降约48-72小时。所有试验在淀粉酶的浓度中均一致，即血清淀粉酶纸将检测到。

就目前而言，DNA是将个人与特定犯罪现场联系起来的最有力的证据。它的特异性毫无疑问，DNA属于谁。犯罪现场的来源是体液的形式：通常是血液或精液，但唾液也带有大量的DNA浓度，因此可能被证明是一项宝贵的证据。但是，与鲜血的颜色和干精液的可见性不同，唾液很难用肉眼看。目前，唾液的检测涉及寻找其主要成分淀粉酶。淀粉酶是唾液中的主要酶，负责消化淀粉。重要的是要有一种检测或绘制唾液的方法，尤其是在受害者口腔袭击的情况下。一种方法是一种交替的光源ALS，它使用导致唾液染色荧光的紫外线，从而增强其可见性。但是，ALS会导致其他体液荧光，而唾液不能与这些[1]区分开，必须使用更具体的测试。另一种方法是使用淀粉酶纸。 The paper is laced with starch; amylase will digest the starch in the paper. When dyed with iodine, the location in which the starch has been digested will appear differently than the rest of the dyed paper. This allows for a mapping of the saliva stain. When mapped, one is then able to make an extract of the saliva and use this to extract DNA. The primary purpose of this study was to assess the sensitivity of the amylase paper by diluting amylase concentrations and testing the paper’s ability to still detect amylase. While part of the study utilized an amylase standard, it is important to note that amylase concentrations in fresh saliva will differ greatly. While the amylase standard, concentrated, contains 1 kU/mL, the average concentration in human saliva is somewhere around 93 IU/mL [2]. Therefore, we also used fresh human saliva to get a more realistic assessment of the sensitivity of the paper.

Materials and methods

•血清淀粉酶纸（德国哥廷根）

• Iodine

• 蒸馏水

•牛仔布样品

• Cotton sample

•聚酯样品

•淀粉酶标准

•盐水溶液（稀释）

Amylase paper was obtained from Seratec (Gottingen, Germany) and stored at room temperature until use. Sections large enough to cover the stained areas were cut out. Fabric, which included cotton, denim, and polyester, was obtained from Jo-Ann Fabrics and washed in a washing machine in cool water, then dried in a dryer at a delicate setting. Amylase standard, catalog number 11111, 1 kU, was obtained from Sirchie (Youngsville, North Carolina). Fresh saliva was collected from five student volunteers, combined, and immediately serially diluted with saline.

Trial 1

1.淀粉酶标准的连续稀释（使用盐水溶液）是由以下浓度的淀粉酶标准制成的：

A。1 kU/1mL

b。500 U/ml

c. 250 U/mL

d。125 U/mL

e. 62.5 U/mL

F。31.25 U/mL

2. Each dilution was added as a stain using 20 microliters to a denim sample, a cotton sample and a polyester sample (Tables 1,2).

3. Stains were mapped over the following time periods:

A。2小时

b。24小时

c. 48 hours

d。1 week

e. 2 weeks

F。3周

Trial 2

1.在此试验中仅使用牛仔布

2. The following dilutions of the amylase standard were used (Table 3)

A。1 k iu

b。500 IU/ML

c. 250 IU/mL

d。125 IU/mL

e. 62.5 IU/mL

F。31.25 IU/mL

g. 15.6 IU/mL

h. 7.8 IU/mL

i. 3.9 IU/mL

j. 2 IU/mL

k. 1 IU/mL

l。0.5 IU/ml

3. The dilutions were added to the denim sample in 30 microliter stains.

4.在以下时间绘制污渍：

A。立即地

b。24小时

c. 48 hours

d。1 week

e. 2 weeks

F。3周

Trial 3

1. This trial involved use of real human saliva samples.

2. 5 samples were collected. 100 microliters from each sample were taken and all were averaged together in a centrifuge tube to account for differences in amylase concentration per person.

3. The following dilutions were made:

A。整洁（350 IU/ml）

b。175 IU/ML

c. 87.5 IU/mL

d。43.75 IU/ml

e. 22 IU/mL

F。11 IU/mL

g. 5.5 IU/mL

h. 2.75 IU/mL

4.污渍在接下来的时间被映射

A。立即地

b。24小时

c. 48 hours

d。72 hours

Upon completion of the first trial, it was evident that the material upon which amylase is deposited will affect the detection of it by amylase paper. Even at equivalent concentrations and time lapsed, there was a difference in detection of amylase. This trial demonstrated that denim is the most effective material in retaining amylase, or at least is the most effective material for a positive test result by amylase paper. Cotton and polyester, on the other hand, did not result in positive test results at lower concentrations or longer amounts of time passed. The cotton material appeared to level off in amylase detection around 250-500 IU/mL. The polyester material appeared to level off around 250-500 IU/mL as well.

The second trial, in which only denim was used once it was determined that denim was the strongest material for amylase detection, demonstrated that detection leveled off around 31.25 IU/mL, around the one week mark. However, this trial had the most sensitive result with a positive test result for amylase at a dilution as weak as 2 IU/mL, when the test was run immediately after depositing on the material.

The third trial, in which the standard was not used but instead saliva from participants, suggests that amylase levels may sharply decline in human saliva around the 24 hour mark. The data demonstrate that the amylase paper detected dilutions as weak as 2.75 IU/mL immediately after the deposit of the saliva onto the material, however, within 24 hours the paper detected a dilution only as weak as 22 IU/mL, evidence for the degradation of amylase in human saliva after 24 hours.

在所有三个试验中，试验3是淀粉酶纸的真实应用中最具代表性的。鉴于在犯罪现场发现的面料上的污渍通常不会是整洁的唾液污渍，因此该试验表明，淀粉酶纸在现场检测唾液的能力和强度最为接近。总之，应注意的是，淀粉酶纸仅检测到在多种体液中发现的淀粉酶，而不是唾液本身。因此，它是一种推定测试，但是Seratec膜技术的本文仍然有用，因为它是便宜的，并且是一种有效的推定测试，可以帮助检测唾液并希望在场景中有用的DNA。

The author would like to thank Dale Laux of Cleveland State University for his providing of materials, support in designing a protocol, and mentorship throughout the project.

1. Mendel AL, Peyrot des Gachons C, Plank KL, Alarcon S, Breslin PAS. Individual differences in AMY1 gene copy number, salivary α-amylase levels, and the perception of oral starch. PLOS ONE. 2010, 5: e13352. PubMed:https://www.ncbi.nlm.nih.gov/pubmed/20967220
2. Virkler K, Lednev I. Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int. 2009, 188: 1-3. PubMed:https://www.ncbi.nlm.nih.gov/pubmed/19328638