A field-deployable automatic nucleic acid extraction and insulated isothermal RT-PCR system for sensitive on-site detection of avian influenza A virus

1. GeneReach Biotechnology Corporation
A field-deployable automatic nucleic acid
extraction and insulated isothermal RT-PCR
system for sensitive on-site detection of
avian influenza A virus
Simon Chung
GeneReach Biotechnology Corp.
Taiwan

2. GeneReach Biotechnology Corporation
2017 New outbreak of avian influenza in China

3. GeneReach Biotechnology Corporation
Present danger to Vietnam
H7N9?

4. GeneReach Biotechnology Corporation
Pen-side / Point of care PCR
• Needs
– Quick results for quick response / control measures / treatment
– Screening test in the field
• Challenge of the current system
– Takes time for transportation of samples from field to lab
• Requirements
– Easy to use: does not require high skill to operate
– Portable equipment (battery operated)
– Easy reagent storage (does not require freezers)
– Cost: inexpensive
– Equivalent level of sensitivity with qPCR in the lab = no compromise

5. GeneReach Biotechnology Corporation
Testing procedure of Pen-side PCR (tacomini and Pockit)
RNA extraction by Taco Mini PCR by Pockit
1. Open cover of pre-loaded plate
2. Add 200ul of sample to the first row
3. Set plate to tacomini and run
4. Wait for 25 minutes
5. Collect 150ul of RNA in elution buffer from the
last row to 1.5 ml tube
1. Take out pre-aliquoted reagent tubes
2. Add 50ul of reaction buffer to each tube
3. Add 5ul of RNA solution
4. Transfer 55ul from reagent tube to reaction tube
5. Set reaction tubes to Pockit and run
6. Wait for 40 minutes
7. Read results (positive / negative)

6. GeneReach Biotechnology Corporation
Materials and methods
• Avian influenza A virus (AIAV)-positive oropharyngeal swab (n =
26) and AIAV-spiked tissue (n = 8; brain, lung and spleen) were
subjected to extraction by tacoTM (tacoTM DNA/RNA Extraction
Kit) and RNeasy Mini Kit (Qiagen) simultaneously. Supernatant
alliquots of tissue homogenate were spiked with serial
dilutions of AIAV H5N1 strain (16A59). Reproducibility was
evaluated by triplicate extractions with 6 tissues and 6 swabs
of various AIAV titers. AIAV RNA from the samples were
quantified by a published qRT-PCR.

7. GeneReach Biotechnology Corporation
Material and methods (cont.)
• Analytical sensitivity of the RT-iiPCR was compared with a qRT-
PCR using an H5N1 isolate (16A59) or H7N9 isolate. The
inclusivity panel included H3, H4, H5, H6, H9 and H10 IAV
subtypes. NA from oropharyngeal swabs and tissue
homogenates (brain, lung and spleen) extracted by
tacoTM were tested to evaluate the RT-iiPCR by comparison
with the qRT-PCR. Interrater agreement was calculated by the
kappa test.

8. GeneReach Biotechnology Corporation
Efficiency of RNA extraction by tacomini in
comparison with QIAGEN RNeasy Mini Kit

9. GeneReach Biotechnology Corporation
Test
Test 1: Test results with H7N9 virus diluted by 10-fold
Virus
dilution
-3 + + + 25.4 25.3 25.4
-4 + + + 28.7 28.6 28.5
-5 + + + 31.8 32.1 32.2
-6 + + + 35.3 35.3 36.2
-7 + + + 40.0 37.0 39.9
-8 + - - neg neg neg
Pockit M qPCR at Lab (Ct value)
Sensitivity and specificity of POCKIT Influenza A reagent for
Influenza A detection
Positive Negative
Pos 23 0 23
Neg 1 18 19
24 18 42
Specificity (%)=
Sensitivity & specificity of Pockit M compared with qPCR M
100.0
94.7
Pockit M
qPCR M*
Total
Total
Sensitivity (%) =

10. GeneReach Biotechnology Corporation
Sensitivity and specificity of POCKIT H7 for H7N9 detection
Test 2: Results of Pockit H7 with samples of Ct value between 27-37
qPCR*
Pos Neg Tested
27 1 0 1 100
28 2 0 2 100
29 2 0 2 100
30 1 0 1 100
31 3 0 3 100
32 2 0 2 100
33 2 0 2 100
34 3 0 3 100
35 3 1 4 75
36 1 0 1 100
37 0 1 1 0
Negative 0 8 8 100
Total 20 10 30
Pockit H7
% Agreement
Number of samples
Ct value
Test
Test 1: Test results with H7N9 virus diluted by 10-fold
Virus
dilution
-3 + + + 25.0 24.8 24.8
-4 + + + 28.2 28.1 28.1
-5 + + + 31.5 32.1 31.7
-6 + + + 35.3 34.8 36.1
-7 + - - 38.6 38.8 37.4
-8 - - - neg neg neg
Pockit H7 qPCR at Lab (Ct value)
Positive Negative
Pos 20 1 21
Neg 0 9 9
20 10 30
Total
Sensitivity (%) = 95.2
Specificity (%)= 100.0
Sensitivity & specificity of Pockit H7 compared with qPCR M
Pockit M
Total
qPCR M*

11. GeneReach Biotechnology Corporation
Pen-side PCR vs qPCR at Laboratory
Test Live bird market / Vet station Laboratory
Step 1:
RNA
extraction
Step 2:
Real-time
PCR
Portable equipment
Easy reagent storage (RT to 4C)
Simple test procedure
Simple result interpretation
Heavy equipment
Reagent stored in freezer
Requires skilled lab operator in
testing and interpretation

12. GeneReach Biotechnology Corporation
Advantages/limitations of tacomini/POCKIT
• Advantages
– Can detect influenza A virus at high sensitivity (100%) and specificity (95%)
– Can detect subtype H7 virus at high sensitivity (95%) and specificity (100%)
– Easy to operate: does not require high skill
– Kit is provided as ready-to-use: less chance of mistake
– Easy to carry: portable equipment, battery operated
– Low cost: POCKIT Micro Plus, tacomini
– Does not require high biosafety when samples are collected to Sample
storage buffer
• Limitations
– POCKIT H7 detect most H7 virus of Eurasian lineage, and not specific to HPAI
H7N9 (AIV HPAI H7 detection reagent has been validated in November)
– It is qualitative (Pos/Neg), not quantitative

13. GeneReach Biotechnology Corporation
Screening Avian Influenza Virus in Northern Vietnam
Photo and edited by Ken Inui

14. GeneReach Biotechnology Corporation
Acknowledgement