Instruction Manual
Table Of Contents
- Intended Use
- Summary and Explanation
- Principles of the Procedure
- Materials Required (Provided)
- DTT solution 500mM
- SARS-CoV-2 PCR Mix
- Equipment and Supplies
- Warnings and Precautions
- Specimen Collection, Handling, and Storage
- Procedure
- For viscous samples (sputum and bronchial wash): Performed inside BSC 2
- NOTE: In the event that the test system becomes inoperable, notify supervision or designee for further direction. Patient specimens must be stored in a manner that maintains the integrity of the specimen.
- Quality Control
- Limitations
- Conditions of Authorization for the Laboratory
- Performance Characteristics
- References
11
Setting Up Real-Time RT-PCR Reactions
1.
In the Reagent Room, thaw two vials of 2019-NCOV PCR MIX for every 96 samples (94 specimens + 2 controls)
in the assay run. DO NOT thaw on a heat block or on the blowers of a biosafety hood.
2. Add 325 µl of 4X TaqPath Enzyme for every 455 µl tube of NCOV PCR MIX. Vortex to mix and spin down.
3.
KEEP 96 WELL PLATE CHILLED ON A COLD BLOCK.
4
To each well of the 96-well reaction plate, add 15 µL of 2019-NCOV PCR MIX as needed. Transfer the 96-
well
Optical Reaction Plate to the PCR Setup Room. Example of an AB7500 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.
Add 10 µL of extracted nucleic acid (from patient specimens and controls) to the appropriate well of the 96-
well
Optical Reaction Plate. Note: Make sure to follow the Reaction Plate tray map.
6.
Cover the plate with Optical Adhesive Cover.
Note: Make sure to handle the Optical Adhesive Cover on the edge only. Do not touch the middle part of the cover.
7.
Briefly centrifuge the plate to collect the reactions at the bottom of the wells and to eliminate any air bubbles.
8.
Take the covered reaction plate to the AB7500.
9
A new window will appear. Select “Absolute quantification” from the “Assay Type” drop-down menu. Select “96-
Well Clear” from the “Container” drop-down menu. Select “COVID RNA TEMPLATE” from the “Template” menu.
Click “Finish” at the bottom of the window.
The 2019-NCOV PCR MIX parameters are:
• NCOV1= FAM
• NCOV3=TAM
• IPC = Q670
10
Real-time RT-PCR parameters using the AB 7500
Stage 1: 50ºC for 15 min
Stage 2: 95ºC for 2 min
Stage 3: 95ºC for 15 sec; 55ºC for 35 sec; 50 cycles.
Sample volume is set at 25 µL
Choose Standard for correct thermal profile parameters.
11
Review the run parameters to make sure they are correct. The passive reference should be set to “ROX.”
12
Go to the Well Inspector Menu (Double-click on 1 of the wells). Highlight the wells not being used and click on
“Omit”. Wells not in use will then contain an X.
13
Refer to PROC.129.028 for details on how to run the plate on the AB 7500 Real Time PCR systems.