YY/T 1876-2023 PDF English
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Tissue engineered medical products - Quantification of remnant DNA in biological materials utilizing animal tissues and their derivatives - Fluorescence method
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YY/T 1876-2023: Tissue engineered medical products - Quantification of remnant DNA in biological materials utilizing animal tissues and their derivatives - Fluorescence method
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/YYT1876-2023
YY
PHARMACEUTICAL INDUSTRY STANDARD
ICS 11.040.30
CCS C 40
Replacing YY/T 0606.25-2014
Tissue engineered medical products - Quantification of
remnant DNA in biological materials utilizing animal tissues
and their derivatives. Fluorescence method
Issued on: JANUARY 13, 2023
Implemented on: JANUARY 15, 2024
Issued by. National Medical Products Administration
Table of Contents
Foreword... 3
Introduction... 5
1 Scope... 7
2 Normative references... 7
3 Terms and definitions... 7
4 Test principle... 9
5 Samples, reagents, and instruments... 9
6 Test procedures... 11
7 Result calculation... 15
8 Result judgment... 17
9 Test report... 18
Bibliography... 19
Tissue engineered medical products - Quantification of
remnant DNA in biological materials utilizing animal tissues
and their derivatives. Fluorescence method
1 Scope
This document specifies a method for the determination of remnant DNA in biological
materials utilizing animal tissues and their derivatives.
This document applies to final products or intermediate products of biological materials
utilizing animal tissues and their derivatives, scaffold materials utilizing animal tissues
and their derivatives used as matrices or scaffolds for tissue engineered medical
products, and can also be used for human-derived decellularized matrix materials.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this
document and are indispensable for its application. For dated references, only the
edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
YY/T 0771.1, Medical devices utilizing animal tissues and their derivatives - Part
1.Application of risk management
YY/T 0771.3, Medical devices utilizing animal tissues and their derivatives - Part
3.Validation of the elimination and/or inactivation of viruses and Transmissible
Spongiform Encephalopathy (TSE) agents
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
extracellular matrix; ECM
Produced by cells and secreted into the extracellular space within tissues, it includes a
homogeneous matrix (proteoglycans and glycoproteins) and filamentous collagen
fibers. It has the function of connecting and supporting cells and is the basic framework
4 Test principle
This document designs a three-step method for the quantitative detection of remnant
DNA in biological materials utilizing animal tissues and their derivatives, namely,
proteinase K digestion and/or tissue homogenization of solid biological materials, DNA
purification, and DNA detection by fluorescent staining.
In addition to the test sample, a spiked recovery test sample must be set up
simultaneously (i.e., a certain amount of DNA control substance is added to the test
sample).
In general, nucleic acid extraction usually uses one or more of the following methods.
cell lysis, tissue homogenization, enzymatic hydrolysis and ultrasonication. In order to
obtain ideal purified DNA, it is recommended to use a combination of enzymatic
hydrolysis and/or tissue homogenization. This combined method is particularly suitable
for situations where decellularized ECM materials are difficult to digest. In order to
avoid the interference of digested decellularized ECM components on DNA detection,
use a nucleic acid extraction kit to purify DNA. In this document, nucleic acid dyes
(such as Picogreen fluorescent dye) are used for the quantitative detection of double-
stranded DNA.
5 Samples, reagents, and instruments
5.1 Test sample
Sample requirements are as follows.
a) Test sample. Decellularized matrix materials (final products or semi-finished
products) and raw materials shall be sterilized and virus inactivated in
accordance with YY/T 0771.1 and YY/T 0771.3 to ensure biosafety;
b) DNA standard curve samples. Use animal cell-derived DNA control samples
to establish a standard curve;
c) DNA purification spike recovery sample. Add a certain amount of DNA
control substance to another sample with the same amount as the sample to be
tested, and use it to determine the spike recovery rate;
d) Negative control sample. Composed of phosphate-buffered saline (PBS) and
bovine serum albumin (BSA), used to exclude contamination during DNA
extraction.
Note 1.For samples whose final products are not easily digested by enzymes
(proteinase K, trypsin, etc.) due to the use of chemical cross-linking and other
5.3.2 Black 96-well plate.
5.3.3 Fluorescence microplate reader.
5.3.4 Low-adhesion DNase-free sterile centrifuge tube.
5.3.5 Low-retention sterile pipette tip.
5.3.6 Refrigerated centrifuge.
5.3.7 Vacuum dryer.
5.3.8 Oscillator.
5.3.9 Vortex mixer.
5.3.10 High-precision balance (0.000 01 g).
5.3.11 Tissue homogenizer.
6 Test procedures
6.1 Sample digestion/homogenization
6.1.1 Preparation of test samples and reaction solutions
The test samples and reaction solutions are prepared as follows.
a) Before sample testing, it is advisable to prepare parallel samples and determine
their moisture content (%) using an appropriate method to calculate the dry
weight of the sample.
b) Accurately weigh the test sample (e.g., 5 mg ~ 10 mg) and record the amount;
place the sample into a 1.5 mL DNase-free sterile centrifuge tube. Take three
parallel samples as the test sample group, and another three parallel samples
as the purified spiked and recovered sample group. The different samples are
processed as follows.
1) Dry sample. Take the sample and weigh it accurately and record it for
subsequent testing.
2) Wet sample. Place the sample in a 1.5 mL DNase-free sterile centrifuge tube
and centrifuge at 15 000 g for 10 min; remove the liquid portion; accurately
weigh, and record the weight for subsequent testing.
3) Colloidal sample. After freeze-drying, accurately weigh and record the
sample; place it in a 1.5 mL DNase-free sterile centrifuge tube. If the
addition of lysis buffer during subsequent DNA purification can decompose
Take 125 μL of the diluted standard solution per well and add it to a 96-well
black ELISA plate, with 3 replicate wells for each sample.
c) Preparation of test samples. Take the purified spiked recovery test DNA
sample and the purified test sample DNA sample; add 1× TE buffer to dilute
appropriately to obtain 400 μL of dilution solution; add 125 μL to each well of
a 96-well black ELISA plate, with 3 replicate wells for each sample.
Note. The dilution factor of the spiked recovery test DNA purification sample
and the test sample DNA purification sample can be adjusted. It is
advisable to ensure that the fluorescence intensity value after the
reaction is within the range of the fluorescence intensity value after the
reaction of the standard solution (less than the fluorescence intensity
value after the reaction of 80 ng/mL).
d) Add 125 μL of PicoGreen reaction solution to each sample above (mixed with
an equal volume of the sample); shake well; incubate at room temperature in
the dark for 5 minutes; measure the fluorescence using a fluorescence
microplate reader. Determination conditions. Use 480 nm as the excitation
wavelength and 520 nm as the emission wavelength for measurement; plot the
obtained data and analyze to obtain the regression equation. Use the
fluorescence intensity value measured with 1× TE buffer as the sample as the
background; measure and record the relative fluorescence intensity (RFI)
value of each measurement well.
Note 1.The DNA content has good linearity in the range of 1.25 ng/mL ~ 80 ng/mL,
and the DNA content of the test sample can be quantitatively determined
within this range; when the DNA content is lower than 1.25 ng/mL, it is a
limit determination, expressed as less than 1.25 ng/mL.
Note 2.PicoGreen reaction solution. Prepare by diluting PicoGreen stock solution
200-fold with 1× TE buffer.
Note 3.1× TE buffer. Prepare by diluting 20× TE buffer 20-fold with DEPC water.
7 Result calculation
7.1 Data processing
First, measure the standard and the sample to be tested 5 times to obtain the relative
fluorescence intensity value; take the average of the relative fluorescence intensity (RFI)
values of the 5 measurements; subtract the background (the RFI value measured when
only TE is present and no DNA is present).
7.2 Calculation of the standard curve equation
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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