WARRANTY

Products are guaranteed to conform to the quality and content indicated on each vial and
external labels during their shelf life. 3B BLACKBIO obligation and purchaser’s rights under this
warranty are limited to the replacement by 3B BLACKBIO of any product that is shown defective
in fabrication, and that must be returned to 3B BLACKBIO, freight prepaid, or at 3B BLACKBIO’
option, replacement of the purchasing price. Any complaint on damaged goods during transport
must be directed to the handling or transport agent.

Manufactured By: TRUPCR® Respiratory Pathogen Panel

Kilpest India Ltd. 7-C Industrial Area, Govindpura, Bhopal-462023 (M.P) Kit
(One Step Real Time PCR Based Detection)
Marketed by:
Version 1.0
Ref Format Content
3B BlackBio Biotech India Limited
7-C Industrial Area, Govindpura, TRUPCR® RESPIRATORY PATHOGEN
Bhopal- 462 023 (M.P.) INDIA 3B273 48 Reactions
PANEL KIT
Tel. (91) 755 4076 518 Store at -20ºC
Fax (91) 755 2580 438
E-mail: info@3bblackbio.com
www.3bblackbio.com

1. DESCRIPTION
TRUPCR® RESPIRATORY PATHOGEN PANEL KIT accurately identified 33 b) Prepare the Reaction mix as follows:
different pathogens which includes Staphylococcus aureus, Streptococcus Name of the Tube Tube Tube Tube Tube Tube Tube Tube
pneumonia, Klebsiella pneumonia, Mycoplasma pneumoniae, Salmonella spp., Reagent 1 2 3 4 5 6 7 8
Streptococcus pyogens, Bordetella spp., Chlamydia pneumoniae, Streptococcus Mastermix-1 10 µl 10 µl 10 µl - - - - -
agalactiae, Acinetobacter baumanii, Pseudomonas aerugenosa, Legionella Mastermix-2 - - - 10 µl 10 µl 10 µl 10 µl 10 µl
pneumonia, Haemophillus influenza A-F, Moraxella catarrhalis., Human Peracho Enzyme Mix 0.35µl 0.35µl 0.35µl 0.35µl 0.35µl 0.35µl 0.35µl 0.35µl
Virus, Human corona Virus (alpha and beta), Parainfluenza 1-4, Influenza A Virus, Primer Probe 9.65
- - - - - - -
Mix-1 µl
Enterovirus, H3N2 Virus, Human Metapneumo Virus (A+B), Pandemic H1 influenza
Primer Probe 9.65
Virus, Influenzae B Virus, Influenzae C Virus, Adeno Virus, Human Respiratory - - - - - - -
Mix-2 µl
Synctial Virus, Human Rhino Virus and Human Boca Virus from respiratory Primer Probe 9.65
specimens (BAL/ Tracheal aspirate/ Sputum/ Nasopharyngeal aspirate in VTM vial) - - - - - - -
Mix-3 µl
using Real time PCR. The human RNaseP gene serves as an endogenous internal Primer Probe 9.65
- - - - - - -
control for human nucleic acid, also included in this kit. Mix-4 µl
Primer Probe 9.65
- - - - - - -
Mix-5 µl
2. REAGENTS INCLUDED IN THE KIT Primer Probe 9.65
- - - - - - -
Mix-6 µl
A. REAGENTS FOR REAL TIME PCR Primer Probe 9.65
Volume in µL - - - - - - -
Reagent Description Mix-7 µl
48 reactions Primer Probe 9.65
Hot-start DNA polymerase - - - - - - -
Mix-8 µl
Reaction Buffer Template 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl
Master Mix-1 dUTPs (dATP, dCTP, 720 µL X 2 (Nucleic acid)
dGTP, dUTP) Total Reaction
MgCl2 and stabilizers 25 µl 25 µl 25 µl 25 µl 25 µl 25 µl 25 µl 25 µl
Volume
Hot-start DNA polymerase
Reaction Buffer c) Transfer 20.0 μl of the above prepared Reaction mix in 0.2 ml PCR tubes
Master Mix-2 dNTPs (dATP, dCTP, 800 µL X 3 and close the tubes.
dGTP, dTTP) d) For 20.0 μl of above reaction mix, add 5.0 µl of extracted nucleic acid
MgCl2 and stabilizers samples or positive control or negative control to make up the final volume
Enzyme Mix Enzyme Mix for RT 67.2 µL X 2 25.0 μl.
Primer Probe Mix-1 231.6 µL X 2
Primer Probe Mix-2 231.6 µL X 2 4. PROGRAM SET UP
Primer Probe Mix-3 231.6 µL X 2 Define the following setting for Temperature Profile
Primer Probe Mix-4 231.6 µL X 2 Dye
Primer
Primer Probe Mix-5 231.6 µL X 2 Step Temperature, °С Time Cycles
Probe Mix Acquisition
Primer Probe Mix-6 231.6 µL X 2 1 37 5 min 1
Primer Probe Mix-7 231.6 µL X 2 2 50 20 min 1
Primer Probe Mix-8 231.6 µL X 2 3 94 10 min 1
Negative Control Sterilized water 500 µL X 2
4 94 10 sec
Positive control for all 8 350 µL X 2 40
Positive control 5 60 60 sec Yes
tubes

5. CHANNEL SELECTION
3. REAL TIME PCR PROTOCOL Define the following setting for channel selection
Tube No Detection Reporter
a) For each sample eight separate reaction mixtures have to make (Tube1, Staphylococcus aureus FAM/Green
Tube 2, Tube 3, Tube 4, Tube 5, Tube 6, Tube 7 and Tube 8). Tube1 for Streptococcus pneumonia HEX/Yellow
Staphylococcus aureus, Streptococcus pneumonia, Klebsiella pneumonia Tube-1
Klebsiella pneumonia Tex Red/Orange
and Mycoplasma spp. detection, Tube 2 for Salmonella spp., Streptococcus
Mycoplasma spp. Cy5/Red
pyogens, Bordetella spp. and Chlamydia spp. detection, Tube 3 for
Salmonella spp. FAM/Green
Streptococcus agalactiae, Acinetobacter baumanii, Pseudomonas
Streptococcus pyogens HEX/Yellow
aerugenosa and Legionella pneumonia detection, Tube 4 for Haemophillus Tube-2
influenza A-F, Moraxella spp., Human Peracho Virus and Human corona Bordetella spp. Tex Red/Orange
Virus (alpha and beta) detection, Tube 5 for Parainfluenza 1-4 detection, Chlamydia spp. Cy5/Red
Tube 6 for type A Influenza Virus, Enterovirus, H3N2 Virus and Human Streptococcus agalactiae FAM/Green
Metapneumo Virus (A+B) detection, Tube 7 for Pandemic H1 influenza Acinetobacter baumanii HEX/Yellow
Tube-3
Virus, Influenzae B Virus, Influenzae C Virus and IC (RnaseP) detection and Pseudomonas aerugenosa Tex Red/Orange
Tube 8 for Adeno Virus, Human Respiratory Synctial Virus, Human Rhino Legionella pneumonia Cy5/Red
Virus and Human Boca Virus detection. Haemophillus influenza FAM/Green
Moraxella spp. HEX/Yellow
Tube-4
Human Peracho Virus Tex Red/Orange
Human corona Virus Cy5/Red
Parainfluenza 1 FAM/Green
Parainfluenza 2 HEX/Yellow
Tube-5
Parainfluenza 3 Tex Red/Orange
Parainfluenza 4 Cy5/Red
type A Influenza Virus FAM/Green
Tube-6
Enterovirus HEX/Yellow
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 H3N2 Virus Tex Red/Orange (RNaseP storage conditions
Human Metapneumo Virus Cy5/Red gene)
Pandemic H1 influenza Virus FAM/Green Nucleic acid of Poor quality
RNaseP HEX/Yellow may interfere with the PCR
The PCR was
Tube-7 reaction, use a
Influenzae B Virus Tex Red/Orange inhibited
recommended isolation
Influenzae C Virus Cy5/Red
method
Adeno Virus FAM/Green
Human Respiratory Synctial Virus HEX/Yellow
Tube-8
Rhino Virus Tex Red/Orange
9. MATERIAL AND DEVICES REQUIRED BUT NOT
Human Boca Virus Cy5/Red PROVIDED
# Gain Setup- auto gain, quencher-none, For Rotor-Gene users, If optimum
fluorescence not generated in auto gain, change to manual gain settings and increase • Adjustable pipettes with sterile filter or positive displacement tips
the gain to desire level • Disposable powder-free gloves
• Sterile bidistilled water
• Sterile 1.5 ml and 2 ml microcentrifuge tubes
6. RESULT ANALYSIS
• Vortex mixer
Tube No
Channels Signal Organism • PCR Machine
• Laminar airflow cabinet
FAM/Green Present Staphylococcus aureus
• PCR vials (0.2 ml, thin‐walled)
HEX/Yellow Present Streptococcus pneumonia
1 • Microtips
Tex Red/Orange Present Klebsiella pneumonia
• 96 – 100% ethanol
Cy5/Red Present Mycoplasma spp.
• Personal protection equipment (lab coat, gloves, goggles)
FAM/Green Present Salmonella spp.
HEX/Yellow Present Streptococcus pyogens
2
Tex Red/Orange Present Bordetella spp. 10. GENERAL PRECAUTIONS
Cy5/Red Present Chlamydia spp.
The user should always pay attention to the following:
FAM/Green Present Streptococcus agalactiae
• Use sterile pipette tips with aerosol barriers and use new tip for every
HEX/Yellow Present Acinetobacter baumanii
3 procedure.
Tex Red/Orange Present Pseudomonas aerugenosa
• Thaw all components thoroughly at room temperature before starting
Cy5/Red Present Legionella pneumonia
detection.
FAM/Green Present Haemophillus influenza
• When thawed, mix the components and centrifuge briefly.
HEX/Yellow Present Moraxella spp.
4 • Use disposable gloves, laboratory coats, and protect eyes while samples
Tex Red/Orange Present Human Peracho Virus and reagents handling. Thoroughly wash hands afterwards.
Cy5/Red Present Human corona Virus • Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in
FAM/Green Present Parainfluenza 1 laboratory work areas.
HEX/Yellow Present Parainfluenza 2 • Do not use a kit after its expiration date.
5
Tex Red/Orange Present Parainfluenza 3 • Dispose of all samples and unused reagents in compliance with local
Cy5/Red Present Parainfluenza 4 authorities’ requirements.
FAM/Green Present type A Influenza Virus • Samples should be considered potentially infectious and handled in a
HEX/Yellow Present Enterovirus biological cabinet in accordance with appropriate biosafety practices.
6
Tex Red/Orange Present H3N2 Virus Infected material and disposable plasticware that was in contact with
Cy5/Red Present Human Metapneumo Virus infected material must be treated with chlorine-containing solutions.
FAM/Green Present Pandemic H1 influenza Virus • Clean and disinfect all sample or reagent spills using a disinfectant, such
HEX/Yellow Present RNaseP as 0.5 % sodium hypochlorite or other suitable disinfectant.
7
Tex Red/Orange Present Influenzae B Virus • Avoid contact with the skin, eyes and mucosa. If skin, eyes and mucosa
Cy5/Red Present Influenzae C Virus contact, immediately flush with water, seek medical attention.
FAM/Green Present Adeno Virus • Material Safety Data Sheets (MSDS) are available on request.
Present Human Respiratory Synctial • Use of this product should be limited to personnel trained in the
HEX/Yellow
8 Virus techniques of DNA/RNA amplification.
Tex Red/Orange Present Rhino Virus
Cy5/Red Present Human Boca Virus The laboratory process must be uni-directional; it should begin in the Extraction
Detection of the Internal Control is not required for positive results. High Bacterial and Area and then move to the Amplification and Detection Areas. Do not return
viral load in the sample can lead to reduced or absent Internal Control signals . samples, equipment and reagents to the area in which the previous step was
performed.
The Cut-off value of this assay is 36 cycles.

7. STORAGE AND HANDLING 11. NOTICE

The user should always pay attention to the following:
All the components of TRUPCR® Respiratory Pathogen Panel Kit should be stored
at -20˚C and stable until the date of expiry stated. The reagents can be aliquoted Store extracted nucleic acid samples at -20°C until ready for use and keep on
and stored at -20˚C in-order to maintain the stability and sensitivity. ice during use.

8. TROUBLESHOOTING IN PCR Avoid microbial contamination of reagents when removing aliquots from reagent
tubes. The use of sterile disposable pipette tips is recommended.
Specimens should be handled as if infectious using safe laboratory procedures.
Comments and Thoroughly clean and disinfect all work surfaces with 0.5% Sodium Hypochlorite in
No. Observation Probable causes
Suggestions de-ionized or distilled water.
Amplification
signal in Cross contamination Check for contamination of
1
negative during handling kit’s component ORDERING INFORMATION
control
Incorrect PCR Check whether all
mixture components are added. Description Format Ref
Missing control
sample during Be careful when pipetting
DNA/RNA mixing TRUPCR® RESPIRATORY PATHOGEN
48 Reactions 3B273
Changing DNA/RNA
Write down sample number PANEL KIT
No on the 1.5 ml micro
during DNA/RNA
amplification centrifuge tube and the PCR
mixing
2 signal with tube
positive Leaving reagents at Please check the storage
controls room temperature condition and the expiration
for a long time or date (see the kit label) of the
incorrect storage reagents and use a new kit,
condition if necessary
The PCR conditions
Repeat the PCR with
do not comply with
corrected settings
the protocol
Weak or no Reagent has been
signal of the thawed and frozen Please mind the storage
3
Internal too often or exposed conditions given in manual
Control to inappropriate

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