Journal: Insects

Article Title: Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

doi: 10.3390/insects14010005

Figure Lengend Snippet: DNA preparation and PCR methods evaluated. Three methods were used to obtain DNA from flea larvae, including a soil DNA isolation kit (S-kit), an ammonium acetate precipitation protocol (AmAcet), and a crude flea lysate protocol. Samples from all extraction protocols were used for PCR amplification using either a hot-start FIREPol ® Taq DNA polymerase or the highly inhibitor-resistant Phusion ® HF DNA polymerase.

Article Snippet: As indicated in , DNA samples obtained using the three DNA preparation protocols were then used for amplification using either a hot-start FIREPol ® Taq DNA polymerase (Solis BioDyne, Tartu, Estonia) or Phusion ® DNA polymerase (New England Biolabs, Ipswich, MA, USA).

Techniques: DNA Extraction, Amplification

Journal: Insects

Article Title: Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

doi: 10.3390/insects14010005

Figure Lengend Snippet: Primer information. Target gene and primer sequences for both forward and reverse primers.

Article Snippet: As indicated in , DNA samples obtained using the three DNA preparation protocols were then used for amplification using either a hot-start FIREPol ® Taq DNA polymerase (Solis BioDyne, Tartu, Estonia) or Phusion ® DNA polymerase (New England Biolabs, Ipswich, MA, USA).

Techniques: Sequencing

Journal: Insects

Article Title: Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

doi: 10.3390/insects14010005

Figure Lengend Snippet: Representative amplification plots for the Tunga penetrans using FIREPol ® Taq ( A ) and Phusion ® ( B ) polymerases. The different DNA preparation methods are color-coded, including pink for AmAcet, blue for S-kit, and black for CL.

Article Snippet: As indicated in , DNA samples obtained using the three DNA preparation protocols were then used for amplification using either a hot-start FIREPol ® Taq DNA polymerase (Solis BioDyne, Tartu, Estonia) or Phusion ® DNA polymerase (New England Biolabs, Ipswich, MA, USA).

Techniques: Amplification

Journal: Insects

Article Title: Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

doi: 10.3390/insects14010005

Figure Lengend Snippet: Success rate for different PCRs and DNA preparation methods based on 30 replicates.

Article Snippet: As indicated in , DNA samples obtained using the three DNA preparation protocols were then used for amplification using either a hot-start FIREPol ® Taq DNA polymerase (Solis BioDyne, Tartu, Estonia) or Phusion ® DNA polymerase (New England Biolabs, Ipswich, MA, USA).

Techniques:

Journal: Insects

Article Title: Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

doi: 10.3390/insects14010005

Figure Lengend Snippet: Comparison of the cycles of quantification (C q value) ( A ) and PCR efficacies ( B ) between different DNA preparation methods and amplification protocols for a Tunga penetrans partial ITS-2 PCR. Template DNA was obtained (i) as a crude lysate (CL) by simple boiling of mechanically cracked larvae, (ii) by proteinase K digestion followed by precipitation with ammonium acetate (AmAcet) or (iii) using a soil DNA isolation kit (S-kit). Amplification was performed as real-time PCR using either the FIREPol ® Taq (Taq) or the Phusion ® (Phu) DNA polymerase protocols. Boxplots show medians with interquartile ranges and whiskers represent 5 and 95% quantiles. Outliers are indicated by dots. The mean of all values is shown as a cross. Paired t -tests for the same template DNA using either Taq or Phusion ® polymerase protocols did not reveal any significant differences. Comparison between different DNA preparation methods using the same amplification protocol were conducted using one-way ANOVAs. Hashtags were used to indicate differences between the DNA preparation methods for the Taq polymerase protocol, while asteriks indicate differences between the preparation methods for the Phusion ® protocol. #, p < 0.05; ***, p < 0.001.

Article Snippet: As indicated in , DNA samples obtained using the three DNA preparation protocols were then used for amplification using either a hot-start FIREPol ® Taq DNA polymerase (Solis BioDyne, Tartu, Estonia) or Phusion ® DNA polymerase (New England Biolabs, Ipswich, MA, USA).

Techniques: Amplification, DNA Extraction, Real-time Polymerase Chain Reaction

Journal: Insects

Article Title: Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

doi: 10.3390/insects14010005

Figure Lengend Snippet: Comparison of the cycles of quantification (C q value) ( A ) and PCR efficacies ( B ) between different DNA preparation methods and amplification protocols for a cox2 partial PCR. Template DNA was obtained as a crude lysate (CL) by simple boiling of mechanically cracked larvae and by proteinase K digestion, followed by precipitation with ammonium acetate (AmAcet) or using a soil DNA isolation kit (S-kit). Amplification was performed as a real-time PCR using either the FIREPol ® Taq (Taq) or the Phusion ® (Phu) DNA polymerase protocols. Boxplots show medians with interquartile ranges and whiskers represent 5 and 95% quantiles. Outliers are indicated by dots. The mean of all the values is shown as a cross. Paired t -tests for the same template DNA using either Taq or Phusion ® polymerase protocols did not reveal any significant differences. Comparisons between different DNA preparation methods using the same amplification protocol were conducted using one-way ANOVAs. # is used to indicate differences between the DNA preparation methods for the Taq polymerase protocol, while * indicates differences between the preparation methods for the Phusion ® protocol. ¤ is used to indicate differences between FIREPol ® Taq and Phusion ® in paired analyses. *, #, p < 0.05; ¤¤, **, p < 0.01; ¤¤¤, ***, ###, p < 0.001.

Article Snippet: As indicated in , DNA samples obtained using the three DNA preparation protocols were then used for amplification using either a hot-start FIREPol ® Taq DNA polymerase (Solis BioDyne, Tartu, Estonia) or Phusion ® DNA polymerase (New England Biolabs, Ipswich, MA, USA).

Techniques: Amplification, DNA Extraction, Real-time Polymerase Chain Reaction