Nucleic acid detection of Plasmodium spp. for dr...
7.1 Principle
The 18ssrRNA gene fragments of Plasmodium spp. in DBS samples are amplified by nested PCR, and then the amplified products are separated by electrophoresis. The species of Plasmodium spp. are determined according to the size in gel by electrophoresis. First of all, the first round of PCR amplification of DNA samples shall be carried out with Plasmodium genus-specific primers (rPLU5/rPLU6 or rPLU7/rPLU8). If the sample DNA contains malaria parasites, the products of about 1,200 bp or 1,000 bp in length can be obtained. Secondly, the species-specific primers of four malaria parasites (Plasmodium falciparum rFAL1/rFAL2, Plasmodium vivax rVIV1/rVIV2, Plasmodium ovale rOAV1/rOAV2, Plasmodium malariae rMAL1/rMAL2 and Plasmodium knowlesi rKNO1/rKNO2) are used for the second round of PCR, and the products with different size could be obtained. According to the size of the products, the species of Plasmodium could be determined: 205 bp for Plasmodium falciparum, 120 bp for Plasmodium vivax, 144 bp for Plasmodium malariae, 800 bp for Plasmodium ovale (Fig. 2-4), Plasmodium knowlesi is about 150 bp.?
Fig. 2-4 Schematic diagram of gene fragments and primers amplified by nest PCR of malaria parasite
7.2 Laboratory layout
The laboratory needs to set up the physical partition according to the functions involved in the reagent preparation area, the sample processing area, the PCR amplification area and the amplification product detection and analysis area. Under suitable condition, each functional area should be set up separately. Otherwise, when the conditions are limited, it can be set up in different areas in the same laboratory. The sufficient room should be retained in different functional area. Furthermore, the amplification product analysis area must be set up independently to avoid contamination to get false positive results (Fig. 2-5).
7.3 Main instruments and equipment
Dry block heaters with shaking function; table-model high speed centrifuge; microcentrifuge; A2 biosafety cabinet/laminar flow clean workbench (optional); two suits of adjustable pipettes : for nucleic acid extraction and PCR, respectively. Each suit includes three pipettes measuring volume in a range of 1-10 μL, 20-100 μL, 100-1,000 μL; special refrigerator: cold layer temperature 2 ℃ ～ 8 ℃, frozen layer -40 ℃ ～ -20 ℃; PCR machine; horizontal electrophoresis apparatus (including glue tray, comb and power supply); microwave oven; gel imaging analyzer; electron balance.
7.4 Main reagent and materials
A. Nucleic acid extraction kit: QIAamp DNA Mini kit (50 cases, Item number: 51304) or others designed for DNA isolation from dry blood spot.
B. Absolute alcohol (analytical purity).
C. Premixed PCR reaction buffer (including Taq plus DNA polymerase, dNTPs, MgCl2, Tris buffer pH 8.3, KCl, bromophenol blue, stabilizer and enhancer, etc.).
D. 50×TBE or 10×TAE buffer.
E. Nucleic acid dyes.
F. DNA marker;
G. Agarose.
H. Clean PCR reaction tube, nuclease free.
I. 1.5 mL eppendorf centrifuge tube;
J. Clean tips with adjustable pipettes.
K. 75% alcohol.
L. Whatman 903 filter paper or other kind of filter paper which can preserve DNA in blood by adding nucleic acid preservation ingredients.
M. Desiccant.
N. Plastic bag.
O. Ultra-fine lancet.
7.5 Preparation and preservation of DBS samples
A. Disinfect the blood collection site with 75% alcohol.
B. After the alcohol dries, an ultra-fine lancet is used to collect blood.
C. Use Whatman 903 filter paper or special blood collection card (adding nucleic acid preservation ingredients) to collect blood, making more than 3 blood spots as possible, each of which is about 1 cm in diameter to ensure that the blood are completely soaked through the filter paper (the blood volume is about 100 μL).
D. Record the sample information on the filter paper and dry naturally in the ventilated place to avoid insects licking and direct sunlight.
E. After completely dry, put the DBS in a plastic bag containing desiccant and store in a dry place. It can be preserved for a long time at -20 ℃ and below.
7.6 Extraction of genomic nucleic acid from DBS samples
The whole genomic DNA of DBS shall be extracted and purified by DNA extraction kits. The operation shall be carried out according to the product instruction. Briefly as follows, take QIAamp DNA Mini kit as an example:
A. Cut 3 spots of DBS in 3 mm diameter into a clean 1.5 mL eppendorf centrifuge tube with a singer-hole paper punch.
B. Add 180 μL ATL buffer, then mix thoroughly by vortexing followed by briefly centrifuge.
C. Add 20 μL pepsin K, votex 20～30 s, briefly centrifuge, incubate at 56 ℃ in a dry block heater for 1h, briefly centrifuge;
D. Add 200 μL AL buffer, then mix thoroughly by votexing followed by briefly centrifuge, incubate at 70 ℃ in a dry block heater for 10 min, briefly centrifuge;
E. Add 200 μL absolute alcohol, mix thoroughly by votexing and briefly centrifuge.
F. Transfer all the liquid in the 1.5 mL eppendorf centrifuge tube to a column, 8,000 rpm/min centrifuge for 1 min, remove the column and put it into a clean 2 mL collection tube.
G. Add 500 μL AW1 buffer to the column, 8,000 rpm/min centrifuge for 1 min, remove the column and put it into a new 2 mL collection tube.(https://www.daowen.com)
H. Add 500 μL AW2 buffer to the column, 14,000 rpm/min centrifuge for 3 min; remove the column and put it into a new 2 mL collection tube, 14,000 rpm/min centrifuge for 1 min.
I. Put the column into a clean 1.5 mL eppendorf centrifuge tube, add 150 μL AE buffer, incubate at room temperature for 1 min and then 8,000 rpm/min centrifuge for 1 min, store the purified DNA at -20 ℃ for use.
7.7 Detection of Plasmodium spp. nucleic acid
7.7.1 Primer sequence for detection of Plasmodium spp. nucleic acid by nested PCR
According to Table 2-1, synthesize the primer sequence needed for the detection of Plasmodium spp. nucleic acid by nested PCR (the purification method shall be PAGE).
Table 2-1 Primers for Detection of Plasmodium Species by Nested PCR and Their Amplified Product Length
7.7.2 Primer dilution
The synthesized primers are centrifuged at 14,000 rpm/min for 3 min. According to the report of primer synthesis, the primer shall be diluted to a stock solution in 100 μmol/L using clean deionized water. Every 10 μL shall be sub-packed and stored at -20 ℃ for use. Before use, the primer stock solution in 100μmol/L shall be diluted to work solution in 10μmol/L by adding 90 μL clean deionized water after dissolving. Repeated freezing and thawing of diluted primer solution should be avoided.
7.7.3 Nested PCR reaction
7.7.3.1 The first round of PCR reaction
PCR reaction system: each reaction system contains 14 μL 2×PCR premixture buffer, 10μmol/L primers rPLU5 and rPLU6 or rPLU7 and rPLU8 0.7 μL each, 1.6 μL DNA extraction as a template, ddH2O brings the total volume to 25 μL.
PCR reaction procedure: 94 ℃ for 3 min, 94 ℃ for 30 s, 58 ℃ for 45 s, 72 ℃ for 60 s. After 34 cycles of steps 2 to 4, 72 ℃ for 6 min.
7.7.3.2 The second round of PCR reaction
PCR reaction system: each reaction system contains 14 μL 2×PCR premixture buffer, 0.7 μL 10 μmol/L primer 1 and 0.7 μL primer 2 (for detection of different malaria parasite species, the primer pairs shown in Table2-1), 2 μL of the products from the first round PCR are used as a template. The total volume shall be brought to 25 μL by ddH2O.
PCR reaction procedure: 94 ℃ for 3 min, 94 ℃ for 30 s, 61 ℃ for 45 s, 72 ℃ for 60 s. After 34 cycles of steps 2 to 4, 72 ℃ for 6 min.
7.7.4 Gel electrophoresis and result analysis
7.7.4.1 Electrophoresis.
A total of 5 μL PCR products from the second round are separated in a 1.5%-2% agarose gel at 120V for 30-40 min. After stained with DNA dye (Goldview or ethidium bromide), the gel is visualized under a gel imaging analysis system.
7.7.4.2 Results Analysis.
The corresponding fragments of 18ssrRNA of four species of malaria parasites are amplified by PCR. The bands of different sizes are shown in the gel after electrophoresis. The results are determined according to the size of the bands. (Fig. 2-6)
Fig. 2-6 Electrophoretic Diagram of Nested PCR Detection of Malaria Parasite
7.8 Quality control of nuclear acid detection of malaria parasite
Monitor the whole process involved in the nuclear acid detection of Plasmodium spp., including the standard operating procedures of nested PCR methods, required instruments and equipment, reagents, materials, the qualification and ability of technicians, and systematically manage the detection activities and report compilation. The objective is to ensure the accuracy, reliability and stability of nucleic acid detection.
The quality control of nucleic acid detection can be divided into internal and external quality control.
7.8.1 Internal quality control
7.8.1.1 Establish the standard operating procedures for the detection of Plasmodium spp. nucleic acid. Ensure that the laboratory technicians strictly follow the standard operating procedures for the detection of Plasmodium nucleic acid.
7.8.1.2 Establish a supplier list of major instruments, equipment, reagents and materials involved in nested PCR. Carry out regular maintenance and qualification of the main instruments and equipment used. Carry out performance qualification for each batch of reagents purchased before use to ensure the effectiveness of the reagents. Establish an log for the use of instruments, materials, and reagents.
7.8.1.3 Establish the log recording of malaria parasite nucleic acid detection, which should include reagent lot number, details of reagent dilution and operation procedure, technicians, test date and so on.
7.8.1.4 Confirm the qualification of the technicians and carry out regular training to ensure that the technicians have the corresponding qualifications and abilities.
7.8.1.5 Carry out the regular proficiency testing for technicians and qualitatively evaluate the ability of technicians to ensure the accuracy of daily test results.
7.8.1.6 In each test, quality control should be set up including positive control (DNA samples containing certain malaria parasites or synthetic plasmids aimed at corresponding amplification fragments), negative control (excluding DNA of malaria parasites to be detected) and blank control (using ddH2O instead of DNA template) to monitor the contamination and validity of the test.
7.8.1.7 Report and feedback of test results.
7.8.1.8 Establish a disposal mechanism for out-of-control of nucleic acid detection. The possible impact factors can be traced and corrected when the false positive, false negative or other errors show during the test.
7.8.2 External quality control
7.8.2.1 Regularly carry out outreach training and supportive supervision (OTSS) activities to evaluate the overall operation of the laboratory and all links. The overall testing ability of the laboratory shall improve through supportive supervision.
7.8.2.2 Participate in regional or national laboratory network, regularly participate in external quality assessment to evaluate laboratory testing ability.
7.8.2.3 Regularly participate in external blind sample testing organized by relevant departments to evaluate the testing ability of technicians.
7.8.2.4 The relevant departments at national or provincial level or a third party shall regularly verify the nucleic acid detection results of the laboratory, randomly pick out the samples already tested in the laboratory in proportion to evaluate the agreement of the test results.