Instruction Manual

7
Procedure
NOTE: For all procedures involving specimens, buttoned lab coats, gloves, and face protection are required
minimum personal protective equipment. Report all accidents to your supervisor and in accordance with the
Company’s policies and Procedures. When performing pooling, laboratories will monitor sample pooling in
accordance with Attachment 1 – Protocol for Monitoring of Sample Pooling Testing Strategies.
SPECIMEN TRANSFER PROCEDURE (to be performed in Extraction Room)
1
Total nucleic acids (DNA and RNA) are extracted from patient specimens and assay controls using the Roche MagNA
Pure 96 DNA and Viral NA Small Volume kit and the Roche MagNA Pure 96 System. Refer to PROC.129.01298-
Nucleic Acid Isolation on the MagNA Pure 96 Instrument for general instructions on using the MagNA Pure 96
Instrument.
2
Decontaminate work area by wiping down work surface and pipettes with 10% bleach. Let soak for 1 minute. Wipe
down work surface and pipettes with 70% ethanol and dry with paper towels.
3
Remove one aliquot of NCOV Positive Control and NCOV Negative Control, from the freezer and let thaw at room
temperature. Vortex and spin down.
4
Create MagNA Pure and AB7500 setup maps for the samples to be tested. 94 patient specimens can be run in a single
set-up, along with 2 controls (1 Positive Control and 1 Negative control per batch). Example of a MagNA Pure set-up
map is provided below
1
2
3
4
5
6
7
8
9
10
11
12
A
POS
8
16
24
32
40
48
56
64
72
80
88
B
1
9
17
25
33
41
49
57
65
73
81
89
C
2
10
18
26
34
42
50
58
66
74
82
90
D
3
11
19
27
35
43
51
59
67
75
83
91
E
4
12
20
28
36
44
52
60
68
76
84
92
F
5
13
21
29
37
45
53
61
69
77
85
93
G
6
14
22
30
38
46
54
62
70
78
86
94
H
7
15
23
31
39
47
55
63
71
79
87
NEG
5
For viscous samples (sputum and bronchial wash): Performed inside BSC 2
a. Add ~ 250 µL of sample to a metal bead tube.
b. Add 350 µL PBS to each tube containing viscous specimen.
c. Vortex tubes for about 20 seconds, repeat if needed.
d. Quick spin to deposit debris in the bottom of the tube.
Alternative method:
a. Rehydrate Thermo Scientific Pierce DTT (Dithiothreitol by adding 100 µ or nuclease- free water to one microtube
containing DTT and gently mix with pipette tip to completely dissolve (500mM final concentration).
b. Add the entire 100 µL of freshly prepared DTT to 5 mL of cold Sterile 0.01M PBS (pH 7.2) and mix briefly.
c. In a microcentrifuge tube, add the diluted DTT solution to an equal volume of specimen (e.g. 250 µL of fresh 500mM
DTT solution to 250 µL of sample). Note: Use DTT immediately. Discard any unused reconstituted DTT.
d. Incubate at room temperature with intermittent mixing until the sample is liquified (up to 30 minutes).
e. Liquefied specimen can be used for downstream nucleic acid extraction.
f.
6
For sample pooling, first pipette 250 µL MagNA Pure External Lysis Buffer into the appropriate well of a MagNA Pure
96 Processing Cartridge. For sample pooling, add equal amounts of each specimen (for 4 specimens, add 50 µL
each) pipette up and down at least once after each addition (performed in BSC 2). The decision to pool specimens
should be based on the positivity rate of the location. Pooling is permitted for NP, OP, AN and MT swabs. For
laboratories considering pooling, please see Attachment 1 for monitoring requirements
7
For controls and all other patient specimens that are not run in a pool, first pipette 250 µL MagNA Pure External Lysis
Buffer into the appropriate well of a MagNA Pure 96 Processing Cartridge. Next, add 200 µL of controls and patient
specimens and pooled specimens, mixing by pipette at least once after each addition (performed in BSC 2).
8
Visually check the level of samples and controls in the MagNA Pure cartridge to ensure sample was added to the
appropriate wells.
9
Cover the MagNA Pure cartridge with an absorbent wipe and put into a clean biohazard bag then seal before
transporting to the MagNA Pure 96 instrument.