COVID-19 Pre-nasal Antigen Test Covid-19 Pre-Nasal Antigen Test,Covid-19 Pre-Nasal Antigen Test Kit,Covid-19 Pre Nasal Test Kit,Quick Test Covid-19 Pre-Nasal Test Weihai Kangzhou Biotechnology Engineering Co.,Ltd , https://www.weihaikangzhou.com
Sharla M. Peters1, Yolanda L. Jones1, Frank Perrella2, Tai Ha3, and Haile F. Yancy1
1U. S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, Maryland 20708, USA. 2U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Compliance, Silver Spring, MD 20993, USA. 3Nebraska Department of Agriculture, 301 Centennial Mall South, Lincoln, Nebraska 68508, USA.
Abstract A real-time fluorescent quantitative PCR technique was developed to detect ruminant source pollution in crude porcine heparin sodium. The assay consists of a set of lyophilized primers, probes and internal controls for ruminants (bovine, sheep, goats) and pigs. The method was validated by two analytical agencies: the first was at the FDA and the second was at an independent laboratory in a state. The performance of heparin sodium multiplex real-time quantitative PCR (hMRTA) detection method passed the strict acceptance criteria of specificity, sensitivity and specificity established by the R&D department of the US FDA Veterinary Drug Center. It meets the sensitivity and repeatability requirements of multiple real-time PCR assays for ruminant raw materials in feeds established earlier. The hMRTA method was used to detect crude porcine heparin sodium, with 98% sensitivity, 98% true positive, and 2% false negative. PCR detection of three ruminant species can be used as a preliminary screening and review confirmation tool for determining the source of pigs. Further ensure the quality of crude heparin sodium and help identify species that control the production of heparin sodium. Controlling the source of crude heparin sodium will ensure the safety of heparin-containing drugs and consumables and protect public health.
An industrial guide (draft) for the quality monitoring of crude heparin sodium is introduced to help pharmaceutical active ingredients, pharmaceuticals, and medical device manufacturers better control the quality of crude heparin sodium used to prevent OSCS or contamination from ruminants. The user is required to establish an appropriate test method or use the USP method to identify and control the crude quality of animal-derived sodium heparin. It has been stipulated in the USP monograph that heparin sodium should clearly indicate the source of the animal species and organ tissue. Drugs and medical device manufacturers that require the purchase of crude heparin sodium should identify the source of each species of crude heparin sodium before producing or preparing to produce heparin-containing drugs or medical devices.
The following assay uses real-time PCR to detect crude heparin sodium ruminant contamination. Reliability was assessed using pig source and bovine source standards. Identify ruminant DNA in crude porcine heparin sodium.
The operating instructions provide specific details of the method, including the reagents and equipment required.
Materials and Methods It is recommended that a negative control be established for each batch of sample to be tested (extraction, purification of impurities and PCR). At the same time, commercial genomic DNA (bovine, pig, goat, sheep) positive controls were set for each batch of BioGX Ruminant and Porcine Beads.
1. Introduction to DNA extraction:
The method for extracting DNA from 0.5 ml of dried dry heparin sodium is described as follows: Please read through the entire standard operating procedure before the extraction operation begins. This method uses the ChargeSwitch® gDNA Rendered Meat Purification Kit manufactured by Invitrogen (Carlsbad, CA). All reagents, tips and tubes are DNase free. Aerosol resistant is required to reduce cross-contamination between samples from DNA extraction to PCR amplification. For handling of powdered substances, it is recommended to wear masks in steps 1, 2 and isolate them from DNA extraction, purification and PCR sites.
Note: PCR detection of crude heparin sodium should be performed prior to any pretreatment (such as chemical oxidation) that affects DNA integrity to avoid affecting PCR species.
1.1. Required materials:
Kit:
ChargeSwitch® gDNA Rendered Meat Purification Kit (Product #CS400-100)
PowerClean® DNA Clean-Up Kit (Product# 12877-50)
BioGX Ruminant and Porcine Beads (Product # 204-0002)
Equipment requirements:
Micro centrifuge
Heat Block
Nuclease Free Water
Vortex
Invitrogen Magna Rack
2.0 mL Tubes
Smart-Cycler
Smart Cycler centrifuge
Smart Cycler cooling block
SmartCycler 25 μL reaction tubes
1.2. Operation steps
a. Add the mixed heparin sodium sample to the 0.5 ml mark line of the 2 ml centrifuge tube.
*Key step: add only one sample at a time, the next centrifuge tube cannot be opened before the previous sample is loaded.
b. Add 1ml of ChargeSwitch Lysis Buffer to the sample. When the lysis buffer was added to the centrifuge tube, the tube was slightly tilted.
c. Add 100 μl of ChargeSwitch SDS to each. Vortex for 5 s to mix.
d. Incubate at 95 ° C (water bath or heating box) for 5 min.
e. Add 400 μl of ChargeSwitch Precipitation Buffer (N5) to each. Vortex for 5 s to mix.
f. The protein was precipitated in an ice bath centrifuge for 5 min.
g. Centrifuge at room temperature 16100g for 5 min.
h. Transfer 1200 μl of the supernatant to a new centrifuge tube.
i. Vortex the centrifuge tube containing the ChargeSwitch Magnetic Beads and resuspend the magnetic beads soaked in the storage buffer.
j. Add 200 μl of ChargeSwitch Detergent to the centrifuge tube containing the supernatant.
k. Add 40 μl of fully resuspended ChargeSwitch Magnetic Beads.
l. Blow up and down 5 times to mix.
m. Incubate for 1 min at room temperature.
n. Place the tube on the MagnaRack until the ChargeSwitch Magnetic Beads form a tight precipitate and the supernatant becomes clear (about
1min).
o. Leave the tube on the magnet and carefully aspirate the supernatant without disturbing the pellet. Do not face the sediment when picking up the supernatant.
p. Remove the centrifuge tube from the magnet.
q. Add 1ml of ChargeSwitch Wash Buffer to the centrifuge tube and mix it up and down 5 times.
r. Place the tube on the MagnaRack until the ChargeSwitch Magnetic Beads form a tight precipitate and the supernatant becomes clear (about
1min).
s. Leave the centrifuge tube on the magnet and carefully aspirate the supernatant without disturbing the sediment. Do not face the sediment when picking up the supernatant.
t. Remove the centrifuge tube from the magnet.
u. Add 750 μl of ChargeSwitch Wash Buffer to the centrifuge tube and mix by pipetting up and down 5 times.
v. Place the tube on the MagnaRack until the ChargeSwitch Magnetic Beads form a tight precipitate and the supernatant becomes clear (about
1min).
w. Leave the centrifuge tube on the magnet and carefully aspirate the supernatant without disturbing the sediment. Do not face the sediment when picking up the supernatant.
x. Add 750 μl of ChargeSwitch Wash Buffer to the centrifuge tube and mix by pipetting up and down 5 times.
y. Place the tube on the MagnaRack until the ChargeSwitch Magnetic Beads form a tight precipitate and the supernatant becomes clear (about
1min).
z. Leave the centrifuge tube on the magnet and carefully aspirate the supernatant without disturbing the sediment. Do not face the sediment when picking up the supernatant. The last time you clean, suck off all the supernatant.
Aa. Remove the centrifuge tube from the magnet and there should be no supernatant in the tube.
Bb. Add 75μl of ChargeSwitch Elution Buffer (E5) to the centrifuge tube.
Cc. Resuspend the ChargeSwitch Magnetic Beads by gently blowing up and down 10 times.
Dd. Incubate for 1 min at room temperature.
Ee. Place the tube on the MagnaRack until the ChargeSwitch Magnetic Beads form a tight precipitate and the supernatant becomes clear (about
1min).
Ff. Leave the tube on the magnet and carefully transfer the supernatant containing the DNA to a new sterile 2 ml centrifuge tube. Do not agitate the pellet. When sucking the supernatant, do not stand against the tip of the gun.
Gg. Discard the used ChargeSwitch Magnetic Beads.
*DNA -20 °C cryopreservation or continue DNA purification to impurities
2. DNA purification to impurities
Introduction:
How to remove the PCR inhibitor (such as heparin) from the above extracted crude heparin sodium DNA is described below. Read through the entire standard operating procedure before purifying impurities. This section uses the PowerClean® DNA Clean-Up Kit from MOBIO (Carlsbad, CA). All kits supplied with the kit are DNase free.
2.1. Operation steps
a. Add 75 μl of nuclease free water to the DNA sample.
b. Add 750 μl of PowerClean DNA Solution 1 to the DNA. Mix upside down 3-5 times.
c. Add 20 μl of PowerClean DNA Solution 2 and mix upside down 3-5 times.
Note: Check PowerClean DNA Solution 2, if there is a precipitate, immerse in a 60 ° C water bath and gently shake until the pellet is completely dissolved. Do not shake it vigorously to avoid a lot of foam. Can be used hot.
d. Add 85 μl of PowerClean DNA Solution 3 and mix upside down 3-5 times. Incubate for 5 min at 4 °C.
e. Centrifuge at 10,000 g for 1 min at room temperature.
f. Avoid sedimentation and transfer all supernatant to a clean 2ml Collection Tube (provided in the kit).
g. Add 70 μl of PowerClean DNA Solution 4 and mix upside down 3-5 times. Incubate for 5 min at 4 °C.
h. Centrifuge at 10,000 g for 1 min at room temperature.
i. Avoid sedimentation and transfer the supernatant to a clean 2ml Collection Tube (provided in the kit).
j. Shake PowerClean DNA Solution 5. Add 800μl of PowerClean DNA Isolation 5 to the supernatant, vortex 5s
Mix well.
k. Load 600 μl of the supernatant into the Spin Filter and centrifuge at 10,000 g for 1 min at room temperature.
l. Discard the filtrate and add the remaining 600 μl of the supernatant to the Spin Filter and centrifuge at 10,000 g for 1 min at room temperature.
m. Discard the filtrate. Add 500 μl of PowerClean DNA Solution 6 to Spin Filer and centrifuge at 10,000 g for 30 s.
n. Discard the filter.
o. Centrifuge at 13,000 g for 2 min at room temperature.
p. Carefully transfer the Spin Filter to a new 2ml Collection tube (provided in the kit). Avoid any PowerClean DNA Isolation 6.
q. Add 75 μl of PowerClean DNA Solution 7 to the center of the white filter.
r. Centrifuge at 10,000 g for 30 s at room temperature.
s. Discard the Spin Filter. Store at -20 °C until PCR amplification.
3. Real-time PCR amplification design This procedure/step is to analyze the presence of ruminant DNA in porcine heparin samples using Cepheid's SmartCycler. The use of this step on other operating platforms requires appropriate adjustments to verify that this part conforms to the standard procedure (corresponding to the platform).
3.1. Preparing for real-time detection
a. Remove the reusable sealed bag containing the sample preparation bead from the refrigerator. The bag is torn from the notch at the top of the sealing area.
b. Remove the required number of tubes from the bag and gently open each tube.
c. Add 25 μL of nucleic acid-free water (no nucleic acid-contaminated water) to each tube, mix with a pipette tip, and cover the tube.
d. Centrifuge all tubes quickly for 5 s using a microcentrifuge.
e. Bead blank control (beads + DNA-free water) must be tested simultaneously with each batch of sample to be tested.
3.2. Steps
a. Dispense 25 μL of the previously prepared master mix (beads and water) into the SmartCycler PCR reaction tube.
b. Add 1 μL of sample to each corresponding PCR reaction tube.
c. Cap the tube and centrifuge for 5 s in a SmartCycler centrifuge.
d. Place the PCR reaction tube into each well of the SmartCycler block and cover each cap.
e. Prepare to run the program:
f. Click the "Create Run" icon. The dye setting selects FCTC.
g. Select the operating procedure (see section 3.3).
h. Select the appropriate number of sites (ie the number of PCR tubes running)
i. Label each site with a sample ID corresponding to each tube component.
j. Click on "Start Run".
Note: A positive result requires a loop threshold (Ct value).
3.3 PCR Reaction Conditions This protocol must be set prior to the start of the PCR run. Save the parameters with a unique name.
PCR program:
The first stage: 95.0 ° C 120s (optical components off)
Second stage: 45 cycles
95.0°C 10s (optical components are off)
56.0°C 60s (optical component on)
Note: When analyzing, the Ct value should be set to the default value of 30.
4. Results Analysis The dye was set to FCTC for fluorescence detection. The positive result of ruminant DNA was judged to be the Ct value of a sample before the 45 reaction cycles of the FAM channel. A positive result for pig material is a positive Ct value in the TxR channel. Internal Amplification Control (IAC) is reported in the Cy5 channel to help reduce false negative reports. Many different types of heparin samples contain inhibitors. This assay includes an IAC to ensure that the PCR conditions are appropriate so that the false negative results reported by the inhibition of the hot start enzyme are minimized. In particular, IAC can reflect negative results reported in the presence of PCR inhibitors in the sample. The primer and probe bind to the sequence synthesized in the reaction mixture.
When there is no PCR inhibitor in the sample and the target is not amplified, the IAC should produce a signal with a Ct value between 32 and 37. If the target sample is at a high concentration, the IAC may or may not report amplification due to competition. this is normal.
If the IAC does not appear at the Ct value of 37, or if it is not reported in the amplification of interest (its Ct value), the sample may contain a PCR inhibitor that prevents the detection of the purpose. If this result is observed, it is recommended to repackage the new crude heparin and use it for additional purification.
For positive results of ruminant samples, the same DNA sample should be additionally tested twice by PCR, ie all three tests are positive, then the sample is confirmed to be positive.
FDA method for detecting crude heparin sodium source
Development of a multiplex real-time PCR assay for the detection of ruminant DNA in raw materials for monitoring the quality of crude heparin sodium