®

Bacterial Meningitis Panel Kit

USER MANUAL

For in vitro Diagnostic Use

Document Code: MB206v1f

Approval Date: 29 June 2016

IVD
 Contents

Page

1. Product Description 1

2. Content 1

3. Storage 1

4. Required Materials and Devices 1

5. Important Notes and Safety Instructions 2

6. Product Use Limitations 2

7. Infection 2

8. Method 3

9. Procedure 3

9.1. Sample Preparation, Storage and Transport 3

9.2. DNA Isolation 3

9.3. Kit Components 4

9.3.1.PCR Master Mix 4

9.3.2.Internal Control 4

9.3.3.Positive Control 4

9.4. Preparing the PCR 5

9.5. Programming the Real-Time PCR Instrument 5

10. Analysis 5

11. Specifications 7

11.1. Sensitivity 7

11.2. Cross Reactivity 7

12. References 7

13. Symbols 7

14. Contact Information 8

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1. PRODUCT DESCRIPTION

Bosphore® Bacterial Meningitis Panel Kit detects and characterizes Haemophilus influenzae, Neisseria
meningitidis and Streptococcus pneumoniae in human biological samples. A region within Haemophilus
influenzae genome is amplified and fluorescence detection is accomplished using the FAM filter. A region
within Neisseria meningitidis genome is amplified and fluorescence detection is accomplished using the HEX
filter. A region within Streptococcus pneumoniae genome is amplified and fluorescence detection is
accomplished using the Texas Red filter.
An internal control has been integrated into the kit in order to check PCR inhibition. The amplification data
of the internal control is detected with the Cy5 filter. The internal control can be added either during DNA
extraction or PCR step.
2. CONTENT
Bosphore® Bacterial Meningitis Panel Kit is composed of Real-Time PCR reagents and positive and
negative controls:

Component REAGENT 100 50 25

Reactions Reactions Reactions
1 dH2O (1000 µl) (1000 µl) (500 µl)
2 PCR Master Mix (1650 µl) (825 µl) (413 µl)
3 Internal Control (550 µl) (275 µl) (138 µl)
4 Positive Control 1 (88 µl) (44 µl) (22 µl)

3. STORAGE
Bosphore® Bacterial Meningitis Panel Kit PCR reagents should be stored at -20°C. Repeated thawing and
freezing (>3x) should be avoided since it may reduce sensitivity. If the components are to be used in small
amounts, they should be frozen in aliquots.
While preparing the PCR; the components should not be exposed to room temperature for more than 10
min. and the detection mix components should not be exposed to light or air more than necessary, vials must
be kept closed except during pipetting. We recommend preparing the PCR on a cooling block and keeping the
detection mixes within a closed container.
The components maintain their stability until the expiry dates on the labels, if they are stored at advised
conditions.

4. REQUIRED MATERIALS AND DEVICES

• Montania® 484 or Montania® 4896 Real-Time PCR Instrument (Anatolia Geneworks), or another
Real-Time PCR system with FAM, HEX, Texas Red and Cy5 filters (iCycler, iQ5, CFX–BioRad,
7500 Real-Time PCR System-ABI, Stratagene Mx3005P, Mx3000P-Agilent, LineGeneK,
LineGene 9600-Bioer, Rotorgene 6000, Q-Qiagen)
• 0.2 ml Thin-Wall PCR tubes, PCR plates or strips
• Magnesia 2448 Nucleic Acid Extraction and PCR Setup Robot and Magnesia 2448 Bacterial DNA
Extraction Kit (Anatolia Geneworks)/ Magnesia® 16 Nucleic Acid Extraction System - Magnesia®
Bacterial DNA Extraction Kit (Anatolia Geneworks) / Magrev® 24 Stand - Magrev® Bacterial DNA
Extraction Kit (Anatolia Geneworks) / Bosphore Bacterial DNA Extraction Spin Kit (Anatolia
Geneworks) or other high quality DNA extraction kits and systems
• Deep freezer (-20°C)

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 • Desktop centrifuge with rotor for 2 ml. microcentrifuge tubes
• Calibrated adjustable micropipettes
• DNAse, RNAse, pyrogen free micropipette tips with filters
• DNAse, RNAse, pyrogen free 1.5 or 2 ml. microcentrifuge tubes
• Disposable laboratory gloves

5. IMPORTANT NOTES AND SAFETY INSTRUCTIONS

Important!:
• The product should be delivered on dry ice. Check for presence of dry ice upon arrival.
• Check for the expiry dates on the box and tube labels, upon arrival. Do not use expired products
or components.
• Calibrated or verified micropipettes, DNAse, RNAse, pyrogen free micropipette tips with filters,
and DNAse, RNAse, pyrogen free microcentrifuge tubes should be used.
• Before starting a test procedure, all components should be thoroughly thawed. After thawing, all
components should be centrifuged briefly (spin-down for 3-5 seconds), and mixed well to ensure
homogeneity prior to use.
• The kit components should be kept on ice or a cooling block until the reaction is prepared, and
they should be quickly returned to -20ºC.
• PCR and nucleic acid isolation must be performed in different compartments. Samples should be
stored separately to avoid contact with the kit components.
• Pathogen information should be reviewed to be aware of the health related risks.
• Biological samples should be handled with extreme caution, suitable class microbiological safety
cabinet should be used: Physical contact with pathogens should be avoided by; wearing lab coats
and gloves, no allowance for eating or drinking within the workspace, prevention of unauthorized
individuals’ access to the working area.
• All the pathogenic wastes produced during the nucleic acid isolation step; including the
serum/plasma samples and material contacted with them, should be discarded into medical waste
and disposed safely.

6. PRODUCT USE LIMITATIONS

• All the components may exclusively be used for in vitro diagnostics.
• This product should be used in accordance with this user manual, by personnel specially trained
to perform in vitro diagnostic procedures.

7. INFECTION
Meningitis is caused by inflammation of the protective membranes covering brain and spinal cord
(meninges). High risk of mortality and morbidity ratios might occur depending on the cause of the infection
such as bacterial, viral agents or use of certain drugs. Viral meningitis is a mild form of disease with higher
incidence compared to acute bacterial meningitis. Acute bacterial meningitis is rarely seen, but it may cause
hearing loss, brain damage and even death if the symptoms emerging instantly cannot be detected in the early
stage.

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8. METHOD
Bosphore Bacterial Meningitis Panel Kit is based on the Real Time PCR method. Polymerase chain
reaction is a technique that is used for amplification of a DNA region. The reaction occurs by the repeating
cycles of heating and cooling. The main components of PCR are primers, dNTPs, Taq polymerase enzyme,
buffer solution and template. As a brief explanation, primers are small synthetic DNA those anneal to the
specific regions of the template in order to start the synthesis. dNTPs are the building blocks of the amplified
products. Taq polymerase amplifies the DNA template. Buffer solution provides the pH adjustment required
for the reaction and template, as referred, is the target region for synthesis.
In Real Time PCR technique, in contrast to conventional PCR, PCR product can be monitored during the
reaction. Therefore Real-Time PCR obviates the need for further analysis methods like gel electrophoresis,
whereby minimizing the risk of contamination. Dual labeled probes employed in the reaction in addition to the
conventional PCR reagents, enable detection of the amplified target with increased sensitivity. I
The assay utilizes the 5’ exonuclease activity of Taq Polymerase to cleave a dual-labeled fluorescent
hydrolysis probe during the extension phase of PCR.
The probe is labeled at the 5’ end with a fluorescent ‘reporter’ molecule, and at the 3’end with another
fluorescent molecule that acts as a ‘quencher’ for the ‘reporter’. When the two fluorophores are in close
proximity, and the reporter is excited by light, no reporter fluorescence can be detected. During the elongation
step of PCR, Taq Polymerase encounters and cleaves the probe bound to the template. As the reporter is
freed from the suppressing effect of the quencher, fluorescence signal can be detected.
The fluorescence generated by the reporter increases as the PCR product is accumulated; the point at
which the signal rises above background level and becomes distinguishable, is called the threshold cycle (C T).
There is a linear relationship between the log of the starting amount of a template and its threshold cycle, thus
starting amount of unknown templates can be determined using standard curves constructed using C T values
of the known starting amounts of target templates.
Bosphore Bacterial Meningitis Panel Kit employs multiplex PCR, and an internal control is incorporated
into the system in order to control the isolation procedure and to check for possible PCR inhibition.
Haemophilus influenzae DNA, Neisseria meningitidis DNA, Streptococcus pneumoniae DNA and an internal
control are co-amplified in a single reaction, using sequence-specific primers. A region within Haemophilus
influenzae genome is amplified and fluorescence detection is accomplished using the FAM filter. A region
within Neisseria meningitidis genome is amplified and fluorescence detection is accomplished using the HEX
filter. A region within Streptococcus pneumoniae genome is amplified and fluorescence detection is
accomplished using the Texas Red filter. The fluorescent signal generated by the internal control amplification,
is detected through the Cy5 channel.
9. PROCEDURE
9.1. Sample Preparation, Storage and Transport
Transportation of the samples should be done according to local and national regulations for pathogen
material transport. Suitable safety and health measures should be taken while working with the pathogens.

9.2. DNA Isolation

We recommend that the Magnesia 2448 Nucleic Acid Extraction and PCR Setup Robot and Magnesia
2448 Bacterial DNA Extraction Kit (Anatolia Geneworks)/ Magnesia® 16 Nucleic Acid Extraction System -
Magnesia® Bacterial DNA Extraction Kit (Anatolia Geneworks) / Magrev® 24 Stand - Magrev® Bacterial DNA
Extraction Kit (Anatolia Geneworks) / Bosphore Bacterial DNA Extraction Spin Kit (Anatolia Geneworks) or
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other high quality DNA extraction kits and systems are used with Bosphore® Bacterial Meningitis Panel Kit.
The DNA isolation should be performed according to the manufacturers’ instructions.
When Magnesia 2448 Nucleic Acid Extraction and PCR Setup Robot is used for DNA extraction, it is
sufficient to start the “Bacterial Meningitis” protocol in the instrument software.
9.3. Kit Components
9.3.1.PCR Master Mix
PCR Master Mix contains a highly specific and accurate HotStarTaq DNA Polymerase, the PCR buffer
and the dNTP Mix. PCR Master Mix also contains forward and reverse primers and dual-labeled probes
specific for Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae genomes and for the
internal control.
9.3.2. Internal Control
An internal control is included in the kit to control RNA isolation and PCR inhibition. The internal control
is a synthetic DNA molecule. It is added into the serum/plasma, proteinase K and carrier RNA mixture during
DNA isolation, to control the isolation efficiency and PCR inhibition. The amount of IC that should be added
during isolation is 5 µl per sample. Alternatively, the internal control can be added directly into the PCR master
mix to control the PCR inhibition exclusively. For this purpose, 0.2 µl of internal control should be added for
each reaction into the master mix. Caution: It is not necessary to include the internal control in the
reaction if it has already been added during the extraction step. Lack of internal control amplification in
the FAM/HEX/Texas RED negative samples, may indicate a problem in isolation or PCR inhibition. In this
case, isolation and PCR should be repeated. In samples that contain a high bacterial load, internal control can
be suppressed and no increase of the signal is detected. Internal control amplification should be evaluated
according to the following table:
Haemophilus Neisseria Streptococcus Internal Result
influenzae meningitidis pneumoniae Control
(FAM) (HEX) (Texas RED) (Cy5)
+ - - +/- Sample is Haemophilus influenzae positive
- + - +/- Sample is Neisseria meningitidis positive
- - + +/- Sample is Streptococcus pneumoniae positive
- - - +/- Sample is negative
+ + - +/- Sample is Haemophilus influenzae and
Neisseria meningitidis positive
- + + +/- Sample is Neisseria meningitidis and
Streptococcus pneumoniae positive
+ - + +/- Sample is Haemophilus influenzae and
Streptococcus pneumoniae positive
+ + + +/- Sample is Haemophilus influenzae, Neisseria
meningitidis and Streptococcus pneumoniae
positive
- - - - Test should be repeated!

9.3.3. Positive Control

The kit includes three synthetic DNA positive controls including:
Positive Control 1: Haemophilus influenzae DNA,
Positive Control 2: Neisseria meningitidis DNA,
Positive Control 3: Streptococcus pneumoniae DNA.
They must be included in every experiment to test the efficiency of the PCR exclusively. The threshold
cycle for the positive control is given in the acceptance criteria table (Section 10. Analysis). Threshold cycles
higher than the acceptance criteria may indicate an efficiency loss in the reaction.

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 9.4. Preparing the PCR
Make sure that all the kit components are thawed before use. Refer to the table below for preparing the
PCR. It is for only one reaction, multiply these values with the sample number to find the values required for
the master mix. While preparing master mixes for more than 5 samples, an extra 10% should be added to the
total sample number.

PCR Master Mix 15 µl

Internal Control * 0.2 µl

Sample DNA 10 µl
(Negative/Positive
Control)

Total Volume 25 µl

* Internal control should not be added in the reaction if it has already been added during the extraction step

Pipette 15 µl of the master mix into the PCR tubes or strips, and add 10 µl of DNA (sample/ positive or
negative control). Close the tube cap. Make sure that the solution in each tube is at the bottom of the tube.
Centrifuge if necessary.
9.5. Programming the Real-Time PCR Instrument
The thermal protocol for Bosphore® Bacterial Meningitis Panel Kit is composed of an initial denaturation
for activation the HotStarTaq DNA Polymerase, a two-step amplification cycle and a terminal hold. The real-
time data is collected at the second step of the amplification cycle.
The thermal protocol to be applied for the reaction is indicated below:
Initial denaturation 95C 14:30 min.

Denaturation 97C 00:30 min.

Annealing 53C 01:30 min. 50 cycles
(Data Collection)

Hold 22C 05:00 min.

Before starting a Real-Time PCR reaction using the Bosphore® Kits, the following steps should be
completed:
• Choose all the filters to be used (FAM, HEX, Texas RED and Cy5),
• Identify unknown samples, positive and negative controls,
• Select the correct thermal protocol.
• Start the experiment.

10. ANALYSIS
By the end of the thermal protocol, the Real-Time PCR Instrument software automatically calculates the
baseline cycles and the threshold.

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 Example of an amplification curve is given in Fig. 1.

Fig. 1: Amplification Curve of a Bosphore® Bacterial Meningitis Panel Kit

Analysis of the results should be performed by trained personnel who have received the required training
for analyzing Real-Time PCR data. We recommend that the test results must be evaluated by an expert
clinician, taking the patient’s clinical findings and the results of other tests into consideration.
All analysis is done automatically in routine use. However, when the trained personnel, who have received
the required training from manufacturer, consider it as necessary, if the system allows pulling down the
threshold as much as possible in order to detect slight amplifications, attention should be paid to keep the
threshold line above the background.
Positive control of Bosphore Bacterial Meningitis Panel Kit is essential for accurate result analysis. The
cycle threshold acceptance criteria for the positive control is 30±3. Test results should not be reported unless
there is amplification of the internal control in negative samples. Please contact the manufacturer if an
impairment in the product’s performance is observed (See the last page for contact information).
The samples that cross the threshold in FAM Channel, HEX Channel, Texas RED Channel and Cy5
Channel are displayed with their positive/negative results, samples that do not cut the threshold are displayed
as “No Ct”. These samples are regarded as negative or having a bacterial load below the detection limit of the
assay. For these undetectable samples, the Cy5 data of the internal control should also be checked to avoid
false negative results.
The table below shows the possible results and their interpretation:
Haemophilus Neisseria Streptococcus Internal Result
influenzae meningitidis pneumoniae Control
(FAM) (HEX) (Texas RED) (Cy5)
+ - - +/- Sample is Haemophilus influenzae positive
- + - +/- Sample is Neisseria meningitidis positive
- - + +/- Sample is Streptococcus pneumoniae positive
- - - +/- Sample is negative
+ + - +/- Sample is Haemophilus influenzae and
Neisseria meningitidis positive
- + + +/- Sample is Neisseria meningitidis and
Streptococcus pneumoniae positive
+ - + +/- Sample is Haemophilus influenzae and
Streptococcus pneumoniae positive
+ + + +/- Sample is Haemophilus influenzae, Neisseria
meningitidis and Streptococcus pneumoniae
positive
- - - - Test should be repeated!

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11. SPECIFICATIONS

11.1. Sensitivity
Analytical sensitivity may be expressed as the limit of detection: i.e. the smallest amount of the target
marker that can be precisely detected. The detection limit of an individual analytical procedure is the lowest
amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value.
The analytical sensitivity or detection limit for NAT assays is expressed by the 95% positive cut-off value.
The analytical detection limit for Bosphore® Bacterial Meningitis Panel Kit was found to 50 copies/ml. The
dilutions were tested in different runs in replicates. The results were analyzed by probit method.

11.2. Cross-Reactivity
To eliminate potential cross-reactivity, both assay design evidence and experimental studies were
employed. Primer and probe sequences were checked for possible homology to other known pathogen
sequences by sequence comparison analysis using database alignment. To eliminate the risk of cross-
reactivity; M. pneumoniae, Brucella, Ureaplasma, M.tuberculosis, K.pneumoniae, S.aureus samples with
known high positivity were tested, and found negative.

12. REFERENCES
1. Tunkel AR. Bacterial Meningitis. Philadelphia: Lippincott Williams & Wilkins, 2001.
2. Tunkel AR, Hartman BJ, Kaplan SL et al. (November 2004). "Practice guidelines for the management
of bacterial meningitis".Clinical Infectious Diseases 39 (9): 1267–84.doi:10.1086/425368.

13. SYMBOLS

Use by

Lot/Batch

REF Catalog number

Temperature limit

Caution, consult accompanying documents

Manufacturer

IVD In Vitro Diagnostic Medical Device

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14. CONTACT INFORMATION

Anatolia Tanı ve Biyoteknoloji A.Ş.

Address: Egitim Mah. Kasap İsmail Sokak No:10/23

34722 - Kadıköy / İstanbul-TÜRKİYE
Tel: +90 216 330 04 55
Faks: +90 216 330 00 42

e-mail: info@ anatoliagenetik.com

www.anatoliagenetik.com

Registered Trademarks: Anatolia Geneworks® Montania®, Magnesia®, Magrev® and Bosphore®

are registered trademarks of Anatolia Tani ve Biyoteknoloji A.S.

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