PHUSA Biochem

SPOT-CHECK
PCR Platform
For CORONA SARS Prevention

Ready-to-use Reagents & Cost-effective Equipment

PHUSA Biochem PCR platform: SPOT-CHECK PCR
Data transfer

Data
analysis

Mobile Device
Data
sharing
IOT

P25 PCR SPOT-CHECK

- LOW COST
- Fast
- No DNA contamination
- Instantly Data Sharing though Internet
SPOT-CHECK based PCR
Genomic
DNA BiH20
PHUSA PCR buffer Stored at
Step 1: PCR mixture preparation
Ready Mix dNTPs
(15-30 mins) RT
Primers
DNA polymerase

Step 2: PCR running (2-3hrs)

Step 3: Data interpretation (10-15 mins)

SPOT-CHECK Mobile device

SPOT-CHECK CONCEPT

It is based on the:
- Melting Temperature of the Amplicons and
the Fluorescence signal emitted by the
complex Amplicons/ Intercalating Dyes
when excited by light.

SPOT-CHECK has 4 components:

Camera
- Blue light source
- Heating Block
- Camera
- Processor

Program Editing SPOT-CHECK

Instrument
SPOT-CHECK CONCEPT

In traditional Fluorescence End-Point PCR, a sample is diagnosed as:

- POSITIVE when get fluorescence
- NEGATIVE when is dark

This concept has problem with false negative samples

that can be cataloged as:
- PCR mix does not contain Primers,
- PCR Rx does not work,
- Extraction DNA does not work,

False Positive samples :

- Primer-Dimer issues.
 SPOT-CHECK CONCEPT

SPOT-CHECK is an enhanced Fluorescence End-Point PCR technique, a sample is diagnosed:

- POSITIVE when get Fluorescence
- NEGATIVE when is dark

To determine which sample is

T1 Picture 1
Positive or Real-Negative, the PCR
samples will go through a series of
heating process with 4 temperatures T2 Picture 2
and at every set temperature, a
picture will be taken to record the
Fluorescence of each sample. The T3 Picture 3
combination of 4 pictures will help
to classify the samples as:
POSITIVE, NEGATIVE, or T4 Picture 4
ERROR (E1, E2, E3).
SPOT-CHECK CONCEPT

- T1/ Pic1: if at T1 (Room Temp), the sample is DARK, the PCR mix does not
contain Primers, we have E1 (Error 1),
- T2/ Pic2: if at T2 (50°C-60°C), the sample is DARK, the PCR reaction didn’t
work, we have E2 (Error 2), our PCR IC fail.
- T3/ Pic 3: if at T3 (70°C-75°C), the sample is DARK, the DNA extraction
didn’t work, we have E3 (Error 3), EXT IC fail
- T4/ Pic 4: if at T4 (80°C-85°C), the sample is DARK, the sample is
NEGATIVE, and POSITIVE if still fluoresce.

In addition to the Temperature/ Picture process, the Primer-Dimer issues

associated with PCR are solved with our PDF-Primers (Primer-Dimer Free)
SPOT-CHECK CONCEPT

T1
Room Temp

Picture 1

E1

At Room Temp, the PCR mix that does not contain Primers
will not Fluoresce, E1.
SPOT-CHECK CONCEPT

T2
50°C-60°C

Picture 2

E1 E0

At 50°C-60°C, the tubes with no PCR Rx will lose Fluorescence, E2,

the PCR IC fail.
SPOT-CHECK CONCEPT

E2

T3
70°C-75°C

E2
Picture 3
E0
E1

At 70°C-75°C, the tubes with no extracted DNA will lose Fluorescence,

E2. The EXT IC fail.
 SPOT-CHECK CONCEPT

E3

T4
80°C-85°C
N

E3
Picture 4
E1
E2

At 80°C-85°C, the tubes with NO DNA target will lose Fluorescence

(NEGATIVE), tubes having DNA target will keep fluoresce (POSITIVE)
SPOT-CHECK CONCEPT

T1 Picture 1 Picture 2 T2

T3 Picture 3 Picture 4
T4
PCR-Analyzer Data Interpreting

A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8
T1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1
T2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1
T3 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1
T4 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0
Result P P P P P P P P P P P E3 E3 E3 E3 E3 E3 E2 E2 E1 N N N N

E3

E3
E1
E2
Traditional PCR Techniques: SPOT-CHECK PCR Platform
(PHUSA Biochem)

3,500 USD
Made locally,
4 hrs process

~15,000 USD ~40,000 USD

4-6 hrs 2-4 hrs
P25 PCR
SPOT-CHECK
Gel Based PCR Real-Time PCR
PDF-PRIMER , Primer-Dimers Free Primer
A universal problem with PCR reactions is the presence of primer-dimers. Primer-
dimers are formed when primers extend each other rather than the target nucleic
acid. Following are some main downfall of primer-dimers
- Primer-dimers use up primers, resulting in the presence of impurities in the
reaction.
- Primer-dimers can use up enough primers to cause false negatives when the
number of copies of target is very LOW.
- Or, if interacting with a probe, primer-dimers can cause false positives.
- In real-time PCR using green dyes (SybrGreen, Evagreen…), Primer-dimers
will give false positives.

A variety of hot starts reagents (active only after being heated at 95*C) have been
developed to deal with the issue of primer-dimers including suspending the
polymerase in a wax material, inhibiting the polymerase with antibodies,
chemically modifying the polymerase, sequestering primers… All those strategies
add more cost and uncertainty to the system.
PDF-PRIMER
To prevent primer-dimers issue, PHUSA Biochem developed a new primers, PDF-
Primers with the 3’OH blocked with a linker (as shown below), and it will be
removed to generate 3’OH when heated at 95*C.

PDF-Primer: Primer-Dimer Free Primer

 PDF-PRIMER

FW: TGCGTCTTCGTTGTTGTTCGCC

RV: TGCGTGTTGCTTGGTTCTTGGC

5’ TGCGTCTTCGTTGTTGTTCGCC 3’
3’ CGGTTCTTGGTTCGTTGTGCGT 5’

Temperature Time Cycle

15 pmol/ primer
room 5min 50ul reaction
95°C 5min 0 0, 1, 10, 100, 1000 copies
95°C 30sec

30sec 35 PCR mix kept for 5 min at room

64oC
72°C 30sec
temperature before PCR
72°C 5min 1
25°C 2min 1
 PDF-PRIMER
1 2 3 4 5
Product

Series 1: 3’OH oligo 100% Primer-Dimer

1 2 3 4 5 Line 1: 0 copy

Series 2: 3’OH oligo 30% Line 2: 1 copy

3’X oligo 70% Line 3: 10 copies

Line 4: 100 copies

Line 5: 1000 copies

Series 1: 3’OH oligo 5% 1 2 3 4 5

3’X oligo 95%
VALIDATION TEST

With the emergence of the Wuhan Corona Virus that

could lead to a major epidemic and health issue, PHUSA
Biochem and Pasteur Institute will joint effort to
demonstrate the validity of PHUSA SPOT-CHECK PCR in
the detection of the CORONA SARS Virus. To do so,
PHUSA Biochem will provide all equipment and reagents
needed. Pasteur Institute will conduct all tests using SPOT-
CHECK PCR in parallel with current WHO guide-line
platform.
VALIDATION TEST Validation experiments

To demonstrate the validity of PHUSA Biochem platform, SPOT-CHECK PCR using

PDF-primers, we will run in parallel the same samples with:
- WHO validated primers, probes, and protocols using qPCR instruments. This run will
be served as reference to evaluate the results of SPOT-CHECK PCR run.
- SPOT-CHECK PCR primers designed using WHO DNA/RNA published sequences of
the CORONA viruses, and the protocols specific to the SPOT-CHECK PCR platform.
- Once the PCR are done, all PCR samples will be analyzed with Agarose Gel
Electrophoresis to confirm the data.

To do the experiments, following are the list of reagents needed:

- RNA materials as template. In the absence of Wuhan Corona viral materials, we will
synthesize all RNA materials needed at PHUSA Biochem lab to make the tests as close
as possible with the reality.
- WHO primers and probes. The primers will be PDF-Primers to allow the use of
normal Taq instead of Hot-Start Taq.
- SPOT-CHECK primers.
VALIDATION TEST REAGENTS

Simple: Just add Extracted DNA then do

Ready-to-use PCR mix packaged
PCR.
in individual PCR tube with
Safe: LESS handling-LESS Contamination.
Optimized/ Customized
Convenient: Ambient temperature shipping,
formulation
storing and handling.

EZ Mix PCR reagent

All oligo will use the The Template will be

WHO Guide-Line used as Positive
sequences Control and to test the
validity of the system.

Oligo Synthetic RNA Template

VALIDATION TEST EQUIPMENT

P25 E100
PCR instrument
Gel Electrophoresis
25 wells
3in1 system

SPOT-CHECK GELPIC 100

Post PCR data analysis Gel Imaging
instrument instrument