PCR Bacteria Test Kit - PromoCell

PCR Bacteria Test Kit 

Instruction Manual

2 Instruction Manual 

Instruction Manual 

3 

Contents 

Reagents and Materials 3 

Application and Test Principle 4 

Precautions 5 

Test Protocol 5 

Appendix 11 

Ordering Information 12 

Reagents and Materials 

Kit Components 

Bacteria Mix red cap; lyophilized 

Containing primer set, deoxynucleotide triphosphates dATP, dCTP, 

dGTP and dUTP, hot-start Taq polymerase and internal amplification 

control; aliquoted for 25 reactions, lyophilized; 1 vial (PK-CA91-2024) 

or 2 vials (PK-CA91-2048) 

Rehydration Buffer blue cap; 1.3 ml 

Positive Control DNA green cap 

DNA-fragment of Bacillus subtilis genome, prepared by PCR, noninfectious, 

lyophilized; 1 vial 

PCR-grade water white cap; 2 ml 


Stability and Storage 

Kit components are stable during shipping. Upon receipt, store at +2 

to +8°C. After rehydration of the Bacteria Mix and the Positive 

Control DNA, store below –18°C and avoid repeated freezing and 

thawing. For repeated testing of low sample numbers Bacteria Mix 

and Positive Control DNA should be aliquoted after rehydration. By 

following these recommendations, the kit is stable until the expiration 

date stated on the label. 

Supplemental Requirements 

• PCR thermal cycler 

• mineral oil, if required for the particular thermal cycler used 

• PCR reaction tubes for the specific PCR device 

• 1.5 ml reaction tubes (DNA and RNA-free) 

• agarose gel electrophoresis apparatus 

• microcentrifuge for 1.5 ml reaction tubes, micropipettes (10, 100 

and 1000 μl) and filter tips



5 

Application and Test Principle 

This kit utilizes the polymerase chain reaction (PCR), thereby 

providing the highest sensitivity for the detection of bacterial 

contamination in cell cultures and other cell culture derived 

biologicals. Detection requires as little as 52 fg of bacterial DNA 

corresponding to 12 bacterial genomes per sample volume. The 

primer set is specific to a highly conserved portion of the 16S rRNA 

coding region in the bacterial genome. The amplified PCR products 

have a size of 466-468 bp (except Micrococcus luteus with 447 bp) 

and can be analyzed by agarose gel electrophoresis. This allows for 

detection of common airborne contaminants in cell cultures including: 

Pseudomonas, Actinomyces, Escherichia, Serratia, Porphyromonas, 

Fusobacteria, Staphylococcus, Streptococcus, Lactobacillus, 

Micrococcus, Bacillus, Klebsiella, Salmonella, Enterococcus, 

Mycobacterium, Legionella, Prevotella, Peptostreptococcus. 

Eukaryotic DNA is not amplified using this kit. Only one protocol is 

needed for the detection of bacteria and can be performed within 3 

hours. 

The kit also provides internal control DNA already included in the 

Bacteria Mix. When running the PCR with the internal control DNA, a 

successful PCR is indicated by a 210 bp band on the agarose gel. 

The lyophilized Bacteria Mix also includes hot-start Taq polymerase as 

well as dATP, dCTP, dGTP and dUTP (instead of dTTP; this provides 

the option to degrade amplicons from previous analyses using uracil- 

DNA glycosylase [UNG]* in order to minimize false-positive results). 

* UNG is not part of the kit. 

This kit is intended for research use only. Not for clinical diagnostics 

or testing of human samples. 

Precautions 

Cross contamination may lead to false-positive results. The test should 

be performed according to good laboratory practice: 

• Clean work surface with plenty of water 

• Always wear gloves 

• Use freshly laundered overalls and protective sleeves 

• Use a facemask and avoid talking during PCR setup 

• Always use new boxes of certified, DNA-free “PCR-clean” 

consumables (tubes, filter tips) 

• Do not autoclave PCR consumables (tubes, filter tips) in 

autoclaves that are also used for autoclaving waste 

• Clean pipettes regularly with plenty of water or DNA-removing 

cleansers 

• Do not work at work benches with recirculated air (laminar flow) 

or badly maintained filters and avoid drafts (e.g., passing 

colleagues) during pipetting 

Test Protocol 

Preparation of Sample Material 

1. Antibiotics can maintain the bacterial contamination at a level 

beneath the detection limit of the test (2.4 x 10 3 bacteria/ml). 

Therefore, prior to testing, the cells should be pre-cultured in the 

absence of antibiotics for at least one passage. 

2. DNA extraction with a commercially available DNA extraction kit 

is always advisable when preparing the samples in order to 

remove inhibitors of the PCR safely and to lyse bacteria 

completely. The obtained DNA extract can be used directly for 

the test avoiding inhibition. 

3. The sample should not contain more than 300 μg/ml DNA. 

4. DNA extracts and heat-inactivated samples can be kept at 2-8°C 

for less than a week and stored at a temperature of at least 

–18°C for a longer period (~1 year; avoid repeated freezing and 

thawing!).



7 

5. The sample should not contain more than 300 μg/ml DNA. 

6. To avoid false positive results, we strongly recommend the use of 

deionized, DNA-free water, aerosol-preventive filter tips and 

gloves. 

Rehydration of the Reagents 

1. Centrifuge tubes with lyophilized components (5 seconds at 

maximum speed of the microcentrifuge) 

2. Bacteria Mix: Add 520 μl of Rehydration Buffer (blue cap) and 

incubate for 5 minutes at room temperature. Then, vortex briefly 

and spin for 5 seconds 

3. Positive Control DNA: Add 300 μl of PCR-grade water (white 

cap) and incubate for 5 minutes at room temperature. Then, 

vortex briefly and spin for 5 seconds 

Keep reagents on ice and store below -18°C after rehydration. Avoid 

repeated freeze-thaw cycles and store reconstituted Positive Control 

DNA in aliquots. 

1. Bacteria Mix, red cap 

Positive Control DNA, green cap 

Spin all freeze-dried components for 5 seconds 

at maximum speed of the centrifuge 

2. Bacteria Mix, red cap Add 520 μl Rehydration BUffer (blue cap) 

3. Positive Control DNA, green cap Add 300 μl PCR grade Water (white cap) 





Incubate 5 minutes at room temperature 

Vortex DNA briefly and spin for 5 seconds 

Test should be performed with negative and positive controls and 

samples in duplicate. All samples and reagents must be equilibrated to 

2-8°C prior to use. 

Loading the test tubes 

Total volume per reaction is 25 μl. When setting up reactions, 

calculations should also include positive and negative controls. 

Aliquot 20 μl of Bacteria Mix into each PCR reaction tube. 

Negative Control: Add 5 μl of PCR-grade water (white cap) to PCR 

reaction tube with 20 μl of Bacteria Mix as negative control. 

Samples: Add 5 μl of sample (cell culture supernatant or DNA extract) 

to PCR reaction tube with 20 μl of Bacteria Mix 

Positive Control: Add 5 μl of Positive Control DNA (green cap) to 

PCR reaction tube with 20 μl of Bacteria Mix 

Close all PCR tubes and spin briefly. Load PCR cycler and start cycler 

program. 

Note: The pipetting sequence should be respected and the tubes 

closed after each sample load. After pipetting the negative control, 

the tube must be sealed before proceeding with the samples. 

Pipetting of the samples and sealing the tubes must also be completed 

before proceeding with the positive control (green cap) in order to 

avoid cross contamination. 

1. Aliquot 20 μl of Bacteria Mix to each PCR tube. 

2. Negative Controls: add 5 μl PCR grade Water or elution buffer from DNA 

extraction kit (ref. chapter “Specimen“) or (white cap). 

3. Samples: add 5 μl of cell culture supernatant or DNA extract. 

4. Positive Control: add 5 μl Positive Control DNA (green cap). 

5. Close and spin all PCR tubes briefly, load the PCR cycler and start the program.



9 

Thermal Profile 

Load the cycler, check each PCR tube and the cycler lid for tight fit. 

Program the PCR cycler or check stored temperature profile. The 

programming process of your cycler is explained in the manual of the 

instrument. 

Program: 

• 1 cycle 

• 94°C for 2 minutes 

• 35 cycles 

• 94°C for 30 seconds 



• cool down to 4°C to 8°C 

Agarose Gel Run 

• 1.5% standard agarose gel, maximal 5 mm thick, with 5 mmcomb 

• load 5 μl of each PCR reaction, mixed with bromophenol blue 

loading buffer per lane (only bromophenol blue in a low 

concentration should be used as a run marker) 

• stop electrophoresis after 2-3 cm run distance (depending on the 

electrophoresis chamber used, e.g. run for 20-30 minutes at 

100 V) 

caused by bacterial DNA loads of >5x10 6 copies/ml. The initial 

concentration of positive control DNA exceeds 5 x 10 6 copies/ml in 

order to account for DNA loss resulting from repeated freezing and 

thawing. Consequently, the internal control is usually not visible in the 

positive control reaction. 

Relevant amplicon sizes: 

Internal control: 210 bp 

Bacteria: 466-468 bp 

Micrococcus luteus: 447 bp 

Results of a successfully performed PCR: 

negative control: band at 210 bp 

positive control/sample: band at 468 bp, possibly additional band 

at 210 bp 

negative sample 

inhibited sample 

positive sample, weak contamination 

positive sample, strong contamination 

negative control with internal control 

1 kb DNA ladder 

Gel Evaluation 

If internal control DNA was used, a distinct 210 bp band should 

appear in every lane indicating a successfully performed PCR. This 

band may fade out with increased amounts of amplicons formed, 

No amplification of Positive Control DNA may be due to the following 

reasons: 

• control DNA tubes have not been spun down before rehydration 

• programming mistake 

• pipetting mistake



11 

Before rerun of a negative and a positive control please check 

thermocycler protocol and pipetting scheme. Interpretation of possible 

band patterns: 

band at 210 bp: 

band at approx. 467 bp and at 210 bp: 

strong band at approx. 467 bp: 

no band: 

Detection of bacteria 

band at approx. 467 bp 

Internal control 

band at 210 bp 

negative sample 

bacteria-positive sample, 

weak contamination 

bacteria-positive sample, 

highly contaminated 

PCR inhibition 

Interpretation 

positive irrelevant bacteria present in the sample 

negative negative PCR inhibition 

negative positive no bacteria detectable in the sample 

2. The reagents supplied should not be mixed with reagents from 

different lots and used as an integral unit. 

3. The reagents of the kit should not be used beyond its shelf life. 

4. For each test setup, at least one negative control should be 

included, possibly reflecting sample preparation. Positive controls 

facilitate the evaluation of the test. 

5. Inhibition may be caused by the sample matrix, but also by 

sample elution buffer of DNA extraction kits not recommended or 

validated. Negative controls should always be completed with the 

use of sample elution buffer. 

6. The use of control samples is advised to secure the day-to-day 

validity of results. The controls should be carried out in the same 

manner as the samples. It is recommended to run laboratory 

specific control samples with a high, medial and low (e.g. 3x 

LOD95) level. Participation in external quality control programs is 

recommended. 

NOTES ON THE TEST PROCEDURE 

Rarely unspecific PCR products can be formed and become visible in 

the gel as faint, diffuse bands of different sizes (not 210 or 467 bp), 

but do not indicate positive results. These faint, diffuse bands are 

mainly caused by non-specific annealing due to overloading of the 

PCR reaction with samples containing more than 300 μg/ml of DNA. 

However, these unspecific PCR products do not indicate positive 

results. 

Possible primer self-annealing produces another band of 80-90 bp in 

length, but also does not affect the precision or results of the test. 

If the PCR of a sample is inhibited (lower band intensity compared to 

negative control), PCR inhibitors can easily be removed from the 

sample by performing a DNA extraction with a commercially available 

kit and testing the sample again. 

1. Read this handbook carefully and follow the recommended 

procedure. Any deviation from the test method can affect the 

results. 

Appendix 

Limited Product Warranty 

This warranty limits our liability for replacement of this product. No 

warranties of any kind, express or implied, including, without 

limitation, implied warranties of merchantability or fitness for a 

particular purpose, are provided. PromoCell shall have no liability for 

any direct, indirect, consequential, or incidental damages arising out 

of the use, the results of use, or the inability to use this product. 

Notice to Purchaser 

This product is optimized for use in the Polymerase Chain Reaction 

(„PCR”) covered by patents owned by Hoffmann-La Roche, Inc., and 

F. Hoffmann-La Roche Ltd. („Roche”). No license under these patents 

to use the PCR process is conveyed expressly or by implication to the 

purchaser of this product. PromoCell does not encourage or support 

the unauthorized or unlicensed use of the PCR process. Use of this 

product is restricted to persons that either have a license to perform 

PCR or are not required to obtain a license.



13 

Ordering Information 

Product Name Size Catalog Number 

PCR Bacteria Test Kit 24 assays PK-CA91-2024 

PCR Bacteria Test Kit 48 assays PK-CA91-2048 

For in vitro research use only. 

Not for diagnostic or therapeutic procedures. 

PromoCell GmbH 

Sickingenstr. 63/65 

69126 Heidelberg 

Germany 

North America 

Phone: 1 – 866 – 251 – 2860 (toll free) 

Fax: 1 – 866 – 827 – 9219 (toll free) 

Deutschland 

Telefon: 0800 – 776 66 23 (gebührenfrei) 

Fax: 0800 – 100 83 06 (gebührenfrei) 

France 

Téléphone: 0800 90 93 32 (ligne verte) 

Téléfax: 0800 90 27 36 (ligne verte) 

United Kingdom 

Phone: 0800 – 96 03 33 (toll free) 

Fax: 0800 – 169 85 54 (toll free) 

Other Countries 

Phone: +49 6221 – 649 34 0 

Fax: +49 6221 – 649 34 40 

Email: info@promokine.info 

05/2013

PCR Bacteria Test Kit - PromoCell

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