Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

BACKGROUND: Detection of bacteria by PCR applied for screening blood and blood products with special attention platelet concentrates. For practical use is expected that the detection system including Gram-positive, Gram-negative and Gram-bacterial non-stainable. It is quite challenging to achieve high sensitivity with clear negative controls with PCR reagents, due mainly Taq DNA polymerase is contaminated with traces of bacteria.

METHODS: Bacterial decontamination of Taq DNA polymerase tested by two different methods using restriction enzymes Sau 3A1 and microfiltration. Besides the commercially available Taq polymerase depleted bacterial DNA was included. A published real-time PCR specific for Gram-negative bacteria adapted to Gram-positive bacteria, including Staphylococcus species specific and Mycobacteria, and is used to fill three Taq polymer-ases discharged from bacteria

RESULTS DNA contamination: Despite published reports of successful DNA decontamination , all three approaches perform poorly in experiments conducted in this study. Sensitivity ranges from about 50-100 colony units (CFU) per PCR reaction to Escherichia coli and Staphylococcus epidermidis, corresponding to 1.250 to 2.500 CFU / ml sample material.

Conclusion: It seems unsatisfactory to receive a high detection limit for the PCR bacteria even if highly multiplexed diagnostics. a reliable method for the decontamination of Taq DNA polymerase is required and will present one important step towards the detection of bacterial DNA with high sensitivity.

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.
Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

Reduce the reaction volume and increase the concentration of Taq DNA polymerase increases STR DNA profile results from the low source Template real world: telogen hair.

OBJECTIVE
The main method for the analysis of low template DNA (LTDNA) is known as a method of low copy number (LCN), which involves increasing the number of PCR cycles (typically 34). In common with other LTDNA method, characterized by an imbalance LCN profile allele, decline, and the drop outs which require interpretation of complex rules. They often require replicate PCR reactions to produce a “consensus” profile in special facilities. The ideal method for the analysis of LTDNA should raise the profile of the results without a high error rate and is accomplished using the standard amenities, in addition to the minimum cost.


METHOD
In this study, we present a comparison of four methods of variation for amplification of STR from LTDNA broadly used, the kit is available commercially (AmpFℓSTR (®) Profiler Plus (®)): the standard method, the standard method to post a PCR clean up, the method of the LCN, and the reaction volume decreases with increased concentrations of Taq DNA polymerase.


RESULTS
Using the telogen hair-LTDNA common source and reference DNA match, LCN method produces the highest number of appropriate and non-appropriate alleles (ie, down-in). For comparison, the reaction volume was reduced by an increase in Taq Polymerase produce fuller profile and corresponding DNA (all alleles combined) and less off-ladder alleles of a variety of input DNA. In addition, this method results in less non-concordant allele of the LCN and not more than for standard PCR, which indicates that it may be preferable to increase PCR cycles for analysis LTDNA, either with or without consensus profiles and statistical modeling.

amfiEco Taq DNA Polymerase, 1 unit/ul,Including dNTP&MgCl2, Bulk Pack

P0701-151 15,000 units
EUR 1560

amfiEco PCR Premix

P0702-096 96 rxns
EUR 186

amfiEco PCR Premix

P0702-480 5x96 rxns
EUR 532.8

amfiEco PCR Premix

P0702-960 10x96 rxns
EUR 984

amfiEco PCR Master Mix,8-Strip RTU(2X), for 50ul reaction

P0704-096 96 rxns
EUR 174

amfiXpand Taq DNA Ploymerase Buffer*(2X)

P0332-001 1ml
EUR 121.2

amfiSure Hot-Start Taq DNA Polymerase

P0322-010 100 units
EUR 176.4

amfiSure Hot-Start Taq DNA Polymerase

P0322-025 250 units
EUR 273.6

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-010 100 units
EUR 180

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl3

P0330-020 2x100 units
EUR 286.8

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-050 5X100 units
EUR 465.6

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-250 25X100 units
EUR 2004

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-500 50X100 units
EUR 3591.6

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-025 250 unit
EUR 200.4

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-050 2X250 unit
EUR 300

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-100 4X250 Unit
EUR 505.2

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-250 10X250 Unit
EUR 886.8

amfiSure ONE Taq DNA Polymerase, 1unit/ul, MgCl2 and dNTP Included

P7002-500 500 units
EUR 262.8

amfiSure ONE Taq DNA Polymerase, 1unit/ul, MgCl2 and dNTP Included

P7002-502 2x500 units
EUR 424.8

amfiSure ONE Taq DNA Polymerase, 1unit/ul, MgCl2 and dNTP Included

P7002-510 10x500 units
EUR 1647.6

amfiSure ONE Taq DNA Polymerase, 1unit/ul, MgCl2 and dNTP Included

P7002-525 25x500 units
EUR 3624

amfiSure ONE Taq DNA Polymerase, 1unit/ul, MgCl2 and dNTP Included

P7002-550 50x500 units
EUR 6459.6

Active Taq Polymerase (Taq)

APX161Ge61 10ug
EUR 280

Eukaryotic Taq Polymerase (Taq)

EPX161Ge61 10ug
EUR 200

Eukaryotic Taq Polymerase (Taq)

RPU59346-100ug 100ug
EUR 672

Eukaryotic Taq Polymerase (Taq)

RPU59346-1mg 1mg
EUR 2925

Eukaryotic Taq Polymerase (Taq)

RPU59346-50ug 50ug
EUR 550

Recombinant Taq Polymerase (Taq)

RPX161Ge01 10ug
EUR 160

Taq

E4A04A03 50ug
EUR 255
Description: Available in various conjugation types.

pTrc- Taq

PVT10500 2 ug
EUR 486

Taq Plasmid

PVT7001 2 ug
EUR 614.4

Taq Polymerase

PCR-211L 5x 200units
EUR 230.7
Description: Thermus aquaticus, recombinant, E. coli

Taq Polymerase

PCR-211S 200units
EUR 57.6
Description: Thermus aquaticus, recombinant, E. coli

Taq Polymerase

PCR-420-10KU 10kilo units
EUR 1043.5

Taq Polymerase

PCR-420-1KU 1kilo unit
EUR 222.3

Taq Polymerase

MBT060-1000U 1 unit
EUR 52.73
Description: Taq Polymerase

Taq Polymerase

MBT060-5000U 1 unit
EUR 204.76
Description: Taq Polymerase

Taq Polymerase

MBT060-500U 1 unit
EUR 35.36
Description: Taq Polymerase

Taq Plus DNA Polymerase (Taq Plus DNA) Protein

abx073522-100tests 100 tests
EUR 225

Taq HS Mix

abx461040-1096tests 10 × 96 tests Ask for price

Taq HS Mix

abx461040-596tests 5 × 96 tests
EUR 1725

Taq HS Mix

abx461040-96tests 96 tests
EUR 475

Taq Plus DNA

enz-309 250U
EUR 60
Description: Recombinant Taq Plus DNA Polymerase

Taq Core Kit

PCR-214L 5 x 200units
EUR 271.9

Taq Core Kit

PCR-214S 200units
EUR 68.2

Green Taq Mix

P131-01 5 ml
EUR 142.8

Green Taq Mix

P131-02 15 ml
EUR 180

Green Taq Mix

P131-03 50 ml
EUR 297.6

10x Taq Buffer

BA00151 1.8ml
EUR 66
Description: High purity buffer for various PCR applications.

Taq DNA Ligase

RK20502 2000U
EUR 105

Taq DNA Polymerase

EQ005 100μl/500U
EUR 50

Taq DNA Polymerase

G009 1000 U (200 ul)
EUR 20
Description: Taq DNA Polymerase is a highly thermostable DNA Polymerase that catalyzes the 5’-3’ synthesis of DNA. This polymerase has 5’-3’ exonuclease activities, lacks 3’-5’ exonuclease activity, and produces 3’-dA-tailed amplicons. PCR products made with Taq can be used with TA cloning vectors.

Taq DNA polymerase

BT10101 500U
EUR 96
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Taq DNA polymerase

BT10102 1000U
EUR 130.8
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Taq DNA polymerase

BT10103 2000U
EUR 200.4
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Taq DNA Polymerase

BA00103 500U
EUR 104.4
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Taq DNA Polymerase

BA00104 1000U
EUR 147.6
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Taq DNA Polymerase

BA00105 2500U
EUR 278.4
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Taq DNA Polymerase

9001-2500 each
EUR 614.4

Taq DNA Polymerase

9001-500 each
EUR 229.2

Taq DNA Polymerase

L7051001 1000 unit
EUR 167

Taq DNA Polymerase

L7051200 200 unit
EUR 43

Taq DNA Polymerase

MB101-0500 500 U/100 μl
EUR 10

Taq DNA Polymerase

abx299725-100g 100 µg Ask for price

Taq DNA Polymerase

abx299725-20g 20 µg
EUR 337.5

Taq DNA Polymerase

abx299725-50g 50 µg Ask for price

Taq DNA Polymerase

abx461029-1096tests 10 × 96 tests
EUR 1787.5

Taq DNA Polymerase

abx461029-596tests 5 × 96 tests
EUR 1062.5

Taq DNA Polymerase

abx461029-96tests 96 tests
EUR 362.5

Champagne Taq antibody

P121-01 500 U
EUR 72.5

Apex Hot Start Taq, 2500u

42-196 5 x 500u/Unit
EUR 572.98
Description: Buffers I & II (Mg Free)

Apex Hot Start Taq, 1000u

42-107 2 x 500u/Unit
EUR 232.71
Description: Buffers I & II (Mg Free)

Apex Hot Start Taq, 5000u

42-210 10 x 500u/Unit
EUR 1075.59
Description: Buffers I & II (Mg Free)

Taq I unit: 410

YRTAQ1 1 vial Ask for price

Taq HS Red Mix

abx461042-1096tests 10 × 96 tests Ask for price

Taq HS Red Mix

abx461042-596tests 5 × 96 tests
EUR 1725

Taq HS Red Mix

abx461042-96tests 96 tests
EUR 475

2 × Taq Master Mix

P111-01 5 ml
EUR 36.91

2 × Taq Master Mix

P111-02 15 ml
EUR 98.42

2 × Taq Master Mix

P111-03 50 ml
EUR 290

Taq Blood PCR Kit

abx461037-1096tests 10 × 96 tests Ask for price

Taq Blood PCR Kit

abx461037-596tests 5 × 96 tests Ask for price

Taq Blood PCR Kit

abx461037-96tests 96 tests
EUR 612.5

Apex Hot Start Taq, 500u

42-106 1 x 500u/Unit
EUR 131.2
Description: Buffers I & II (Mg Free)

Apex Hot Start Taq, 10,000u

42-211 20 x 500u/Unit
EUR 2162.67
Description: Buffers I & II (Mg Free)

Apex Hot Start Taq, 50,000u

42-212 100 x 500u/Unit
EUR 8349.64
Description: Buffers I & II (Mg Free)

Apex Taq, 500Units, 5u/ul

42-800B1 1 x 500u/Unit
EUR 65.71
Description: 10X Standard & NH4 Buffers

Apex Taq, 500Units, 5u/ul

42-800B2 1 x 500u/Unit
EUR 64.49
Description: 10X Standard MgFree&NH4 MgFree

Apex Taq, 500Units, 5u/ul

42-800B3 1 x 500u/Unit
EUR 82.81
Description: 10X Combination Buffer

Apex Taq, 500Units, 5u/ul

42-800B4 1 x 500u/Unit
EUR 63.05
Description: Glycerol Free; No Rxn Buffer

Apex Taq, 1,000Units, 5u/ul

42-801B1 2 x 500u/Unit
EUR 126.43
Description: 10X Standard & NH4 Buffers

Apex Taq, 1,000Units, 5u/ul

42-801B2 2 x 500u/Unit
EUR 129.98
Description: 10X Standard MgFree&NH4 MgFree

Apex Taq, 1,000Units, 5u/ul

42-801B3 2 x 500u/Unit
EUR 132.2
Description: 10X Combination Buffer

Apex Taq, 1,000Units, 5u/ul

42-801B4 2 x 500u/Unit
EUR 134.14
Description: Glycerol Free; No Rxn Buffer

Apex Taq, 5,000Units, 5u/ul

42-802B1 10 x 500u/Unit
EUR 644.58
Description: 10X Standard & NH4 Buffers

Apex Taq, 5,000Units, 5u/ul

42-802B2 10 x 500u/Unit
EUR 555.22
Description: 10X Standard MgFree&NH4 MgFree

Apex Taq, 5,000Units, 5u/ul

42-802B3 10 x 500u/Unit
EUR 549.95
Description: 10X Combination Buffer

Apex Taq, 5,000Units, 5u/ul

42-802B4 10 x 500u/Unit
EUR 536.63
Description: Glycerol Free; No Rxn Buffer

Apex Taq, 10,000Units, 5u/ul

42-803B1 20 x 500u/Unit
EUR 1219.22
Description: 10X Standard & NH4 Buffers

Apex Taq, 10,000Units, 5u/ul

42-803B2 20 x 500u/Unit
EUR 1253.36
Description: 10X Standard MgFree&NH4 MgFree

Apex Taq, 10,000Units, 5u/ul

42-803B3 20 x 500u/Unit
EUR 1428.96
Description: 10X Combination Buffer

Apex Taq, 10,000Units, 5u/ul

42-803B4 20 x 500u/Unit
EUR 1182.48
Description: Glycerol Free; No Rxn Buffer

2×Taq PCR Mixture

E28IT0048 5×1ml
EUR 50.79

Ruby Taq Master (2x)

PCR-164L 20 x 1.25ml
EUR 466.7
Description: Ready-to-Use Mixes for PCR

Ruby Taq Master (2x)

PCR-164S 4 x 1,25ml
EUR 116.6
Description: Ready-to-Use Mixes for PCR

Ruby Taq Master (2x)

PCR-164XL 100ml
EUR 1492.9
Description: Ready-to-Use Mixes for PCR

Taq 2X PCR MasterMix

G888 400 rxn (10.0 ml)
EUR 60
Description: Taq 2X PCR MasterMix is a ready-to-use MasterMix containing Taq DNA Polymerase (Cat. No. G009) in a uniquely-formulated buffer with gel loading dye. Taq has 5’-3’ polymerase and 5’-3’ exonuclease activities, lacks 3’-5’ exonuclease activity, and produces 3’-dA-tailed amplicons. PCR products made with Taq can be used with TA cloning vectors.

2X PCR Taq MasterMix

G900 25.0 ml (1000 Rxns)
EUR 246

2X PCR Taq MasterMix

G013 5.0 ml (200 Rxns)
EUR 104.4

Wise Taq Premix (96Rx)

9K-002-0028 96Rx, 96prep
EUR 283.94

Wise Taq Premix (576Rx)

9K-002-0032 576Rx, 576prep
EUR 1066.94

10x Taq Buffer (MgCl2)

PCRB16 4x1.5ml, 6ml
EUR 70.44

Taq Polymerase (5U/µl)

MBT060A-1000U 1 unit
EUR 62.03
Description: Taq Polymerase (5U/µl)

Taq Polymerase (5U/µl)

MBT060A-5000U 1 unit
EUR 241.01
Description: Taq Polymerase (5U/µl)

Taq Polymerase (5U/µl)

MBT060A-500U 1 unit
EUR 41.58
Description: Taq Polymerase (5U/µl)

Taq Polymerase(3U/µl)

MBT060B-1000U 1 unit
EUR 73.68
Description: Taq Polymerase(3U/µl)

Taq Polymerase(3U/µl)

MBT060B-5000U 1 unit
EUR 286.42
Description: Taq Polymerase(3U/µl)

Taq Polymerase(3U/µl)

MBT060B-500U 1 unit
EUR 49.43
Description: Taq Polymerase(3U/µl)

Taq Polymerase(3U/µl)

MBT060C-1000U 1 unit
EUR 73.68
Description: Taq Polymerase(3U/µl)

Taq Polymerase(3U/µl)

MBT060C-5000U 1 unit
EUR 286.42
Description: Taq Polymerase(3U/µl)

Taq Polymerase(3U/µl)

MBT060C-500U 1 unit
EUR 49.43
Description: Taq Polymerase(3U/µl)

Taq Polymerase(1U/µl)

MBT060D-1000U 1 unit
EUR 73.68
Description: Taq Polymerase(1U/µl)

Taq Polymerase(1U/µl)

MBT060D-5000U 1 unit
EUR 286.42
Description: Taq Polymerase(1U/µl)

Taq Polymerase(1U/µl)

MBT060D-500U 1 unit
EUR 49.43
Description: Taq Polymerase(1U/µl)

Taq Polymerase(1U/µl)

MBT060E-1000U 1 unit
EUR 73.68
Description: Taq Polymerase(1U/µl)

Taq Polymerase(1U/µl)

MBT060E-5000U 1 unit
EUR 286.42
Description: Taq Polymerase(1U/µl)

Taq Polymerase(1U/µl)

MBT060E-500U 1 unit
EUR 49.43
Description: Taq Polymerase(1U/µl)

TM Taq DNA Polymerase

TBS4010 1000 U
EUR 60

Crystal Taq Master (2x)

PCR-166L 20 x 1.25ml
EUR 466.7
Description: Ready-to-Use Mixes for PCR

Crystal Taq Master (2x)

PCR-166S 4 x 1,25ml
EUR 116.6
Description: Ready-to-Use Mixes for PCR

Crystal Taq Master (2x)

PCR-166XL 100ml
EUR 1492.9
Description: Ready-to-Use Mixes for PCR

Taq HS DNA Polymerase

P132-d1 500 U
EUR 47.85

Taq HS DNA Polymerase

P132-d2 1,000 U
EUR 89.18

Taq HS DNA Polymerase

P132-d3 5,000 U
EUR 406

Taq DNA Polymerase (PCR)

G008 5000 U (1.0 ml)
EUR 246

Taq DNA Polymerase (PCR)

G126 10000 U (2 x 1.0 ml)
EUR 370.8

Taq DNA Polymerase (500U)

9K-001-0001 500U
EUR 209.29

Taq DNA Polymerase (3000U)

9K-001-0002 3000U
EUR 582

Taq DNA Polymerase (6000U)

9K-001-0033 6000U
EUR 1234.5

Taq DNA Polymerase (1000U)

9K-001-0035 1000U
EUR 418.62

Taq DNA Polymerase Mix

abx461043-1096tests 10 × 96 tests Ask for price

Taq DNA Polymerase Mix

abx461043-596tests 5 × 96 tests
EUR 1062.5

Taq DNA Polymerase Mix

abx461043-96tests 96 tests
EUR 337.5

Taq Reaction Buffer 10X

11170008-1 5 mL Ask for price

Taq Plus DNA Polymerase

P201-01 250 U
EUR 147.6

Taq Plus DNA Polymerase

P201-02 1000 U
EUR 213.6

Taq Plus DNA Polymerase

P201-03 3000 U
EUR 382.8

Taq Reaction Buffer - Red

abx461046-1096tests 10 × 96 tests Ask for price

Taq Reaction Buffer - Red

abx461046-596tests 5 × 96 tests Ask for price

Taq Reaction Buffer - Red

abx461046-96tests 96 tests
EUR 500

Taq DNA polymerase (-dNTPs)

02-011 200U
EUR 168
Description: The Taq DNA polymerase (-dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (-dNTPs)

02-011-5 5×200U
EUR 261.6
Description: The Taq DNA polymerase (-dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (+dNTPs)

02-001 200U
EUR 183.6
Description: The Taq DNA polymerase (+dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (+dNTPs)

02-001-5 5×200U
EUR 308.4
Description: The Taq DNA polymerase (+dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq Polymerase - high conc.

PCR-421-100KU 100kilo units
EUR 5843.6

Taq Polymerase - high conc.

PCR-421-10KU 10kilo units
EUR 1168.7

Taq Polymerase - high conc.

PCR-421-1KU 1kilo unit
EUR 248.9

Taq DNA Polymerase (TaqDNA) Protein

abx073521-100tests 100 tests
EUR 225

Taq DNA Polymerase, 5units/㎕

T2200-050 500units
EUR 157.2

Taq DNA Polymerase, 5units/㎕

T2200-100 2x500 units
EUR 230.4

Taq DNA Polymerase, 5units/㎕

T2200-200 4x500 units
EUR 375.6

Maximo Taq DNA Polymerase

S101 500 units
EUR 55.2

Maximo Taq DNA Polymerase

S102 5x500 units
EUR 128.4

Maximo Taq DNA Polymerase

S103 20x500 units
EUR 393.6

EZ Taq Polymerase (HotStart)

9K-005-0020 500U
EUR 245.83

Hotstart Taq DNA Polymerase

L7052001 1000 units
EUR 595

Hotstart Taq DNA Polymerase

L7052250 250 units
EUR 167

Champagne Taq DNA Polymerase

P122-d1 500 U (2.5 U/μl)
EUR 212.4

Champagne Taq DNA Polymerase

P122-d2 500 U (5 U/μl)
EUR 212.4

Champagne Taq DNA Polymerase

P122-d3 500 U (10 U/μl)
EUR 212.4

Accuris Hot Start Taq, 1000 units

PR1000-HS-1000 1 PC
EUR 383.06

Accuris Hot Start Taq, 6000 units

PR1000-HS-6000 1 PC
EUR 1848.75

Taq Reaction Buffer - Colorless

abx461045-1096tests 10 × 96 tests Ask for price

Taq Reaction Buffer - Colorless

abx461045-596tests 5 × 96 tests Ask for price

Taq Reaction Buffer - Colorless

abx461045-96tests 96 tests
EUR 437.5


CONCLUSION

Overall, this study highlights the importance and benefits of optimizing PCR conditions and to develop improved laboratory method to amplify and analyze LTDNA.