J Infect Dis. Simplified hot start PCR. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method.
J Clin Microbiol. Highly sensitive single-step PCR protocol for diagnosis and monitoring of human cytomegalovirus infection in renal transplant recipients. J Virol Methods. Viral diagnosis of neurological infection by RT multiplex PCR: a search for entero- and herpesviruses in a prospective study. Cassinotti P, Siegl G. Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections. Specificity, efficiency, and fidelity of PCR.
PCR Methods Appl. Optimization of multiplex PCRs. The polymerase chain reaction. Boston, Mass: Birkhauser; Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. PCR protocols: a guide to methods and applications. San Diego, Calif: Academic Press; Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.
Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Herpes simplex virus infections of the central nervous system in human immunodeficiency virus-infected patients: clinical management by polymerase chain reaction assay of cerebrospinal fluid. Clin Infect Dis. J Clin Pathol. Della N G. Molecular biology in ophthalmology: a review of principles and recent advances. Arch Ophthalmol. The clinical diagnosis of genital ulcer disease in men.
General concepts for PCR primer design. Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR. Multiplex PCR: advantages, development, and applications. Reverse transcription multiplex PCR for differentiation between polio and enteroviruses from clinical and environmental samples. Diagnosis of viral chlamydial keratoconjunctivitis: which laboratory test? Br J Ophthalmol. Emmanuel P J. Polymerase chain reaction from bench to bedside.
Applications for infectious disease. J Fla Med Assoc. Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction enzyme hybridization assay Hexaplex Clin Infect Dis. Rapid detection of Actinobacillus actinomycetemcomitans , Prevotella intermedia and Porphyromona gingivalis by multiplex PCR. J Periodontal Res.
Detection and identification of human papilloma viral DNA, types 16, 18, and 33 by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay.
Rapid identification of nine microorganisms causing acute respiratory tract infection by single-tube multiplex reverse transcription-PCR: feasibility study. Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction.
Mol Cell Probes. Harris E. Appropriate transfer of molecular technology. Asia Pacific Technol Monitor. Harrison T J. The polymerase chain reaction—a time of transition from research to routine. Transcription of the human cytomegalovirus natural killer decoy gene, UL18, in vitro and in vivo.
J Gen Virol. Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions. Multiplex PCR: critical parameters and step-by-step protocol. Hengen P N. Trends Biol Sci.
Clin Diagn Virol. Co-amplification of multiple regions of the HIV-1 genome by the polymerase chain reaction: potential use in multiple diagnosis. Jackson J B. Polymerase chain reaction in transfusion medicine. Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs.
Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses. Development of a dual target-PCR for detection and characterization of measles virus in clinical specimens. Kaucner C, Stinear T. Sensitive and rapid detection of viable Giardia cysts and Cryptosporidium parvum oocysts in large-volume water samples with wound fiberglass cartridge filters and reverse transcription-PCR. App Environ Microbiol. Multicenter international work flow study of an automated polymerase chain reaction instrument.
Clin Chem. Kwok S, Higuchi R. Avoiding false positives with PCR. Lazarus P, Caruana S. Typing of common human papilloma virus strains by multiplex PCR. Anal Biochem. Systematic studies on parameters influencing the performance of the polymerase chain reaction.
J Clin Chem Clin Biochem. Conducting polymers on microelectronic devices as tools for biological analyses. Clin Chim Acta. Detection of Chlamydia trachomatis , Neisseria gonorrhoeae , Ureaplasma urealyticum , and Mycoplasma genitalium in first-void urine specimens by multiplex polymerase chain reaction.
Mol Diagn. Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genitourinary specimens. Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection. J Clin Lab Anal. Amplification of the six major human herpesviruses from cerebrospinal fluid by a single PCR. Simultaneous detection of three common sexually transmitted agents by polymerase chain reaction.
Am J Obstet Gynecol. Mitsuhashi M. Technical report: part 2. Basic requirements for designing optimal PCR primers. Comparison of clinical diagnosis and standard laboratory and molecular methods for the diagnosis of genital ulcer disease in Lesotho: association with human immunodeficiency virus infection.
Detection of human herpesvirus 6 and 7 in heart transplant recipients by multiplex polymerase chain reaction method. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies. Diagnosis of cytomegalovirus pneumonia by polymerase chain reaction with archived frozen lung tissue and bronchoalveolar lavage fluid.
Am J Clin Pathol. The design of strain-specific polymerase chain reaction for discrimination of the raccoon rabies virus strain from indigenous rabies viruses of Ontario. Simultaneous amplification and detection of specific hepatitis B virus and hepatitis C virus genomic sequences in serum samples. Selecting optimal oligonucleotide primers for multiplex PCR. Intelligent Syst Mol Biol. Simultaneous PCR detection of Haemophilus ducreyi , Treponema pallidum , and herpes simplex virus types 1 and 2 from genital ulcers.
Osiowy C. Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-PCR assay. Simultaneous polymerase chain reaction detection and restriction typing for the diagnosis of human genital papillomavirus infection. Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol. Pozo F, Tenorio A. Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction.
Multiplex polymerase chain reaction for subgenus-specific detection of human adenovirus in clinical samples. Raeymaekers L. A commentary on the practical applications of competitive PCR.
Genome Res. Rajeev B, Biswas J. Molecular biologic techniques in ophthalmic pathology. Indian J Ophthalmol. Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay.
Aseptic meningitis and encephalitis: the role of PCR in the diagnostic laboratory. Betaine can eliminate the base pair composition dependence of DNA melting.
Dilution of the environmental sample restored the parallelism of amplification curves to that of standard, suggesting the inhibition was marginal in this specific case. In addition to BSA, we further tested chemical and protein additives on the real time PCR reaction containing inhibitor-rich samples.
For the nature of the PCR inhibitor from our sampling site, chemical additives did not improve the PCR reaction further. However, addition of GP protein further recused the PCR reaction and it lessened the decrease in saturated fluorescence among the tested additives and generated a PCR amplification curve with a slope similar to that of standard Table 1.
This preliminary result prompts to further tests of PCR additives for tackling inhibitor-rich environmental samples in the future. At present, the site-specific PCR inhibitor issue was not completely solved and PowerClean purification offers an option to remove the inhibitors despite of its variable and low purification efficiency.
We anticipated that water samples from the coastal area are highly contaminated and should be treated by PowerClean purification before qPCR. However, water samples away from shores may contain less PCR inhibitors, and thus PowerClean purification can be removed from the standard procedure to reduce cost and loss in eDNA contents. Besides testing the efficiencies, we also improved the extraction workflow by introducing a spin column that fits into a 50 mL centrifuge tube for collecting the lysate from the Sterivex cartridge.
The use of the spin column along with a swing-type rotor allows a higher recovery of lysate, as some lysate often remains in the Sterivex cartridge when centrifuged with an angled rotor Fig. A larger volume of lysate can be accommodated by the 50 mL centrifuge tube, which allows a greater choice of the volume of initial digestion.
When using parafilm to plug the inlet or outlet, liquid often leaks during incubation and handling, which causes cross contamination and loss of samples. The introduction of stoppers and silicon tube connections improved the extraction procedures and decreased the contamination chances. These improvements together increase the throughput and decrease the labor force for extraction. In conclusion, we optimized a new protocol for the extraction of eDNA from Sterivex cartridge and for quantification of specific species using real time PCR, focusing on the quantitative aspects.
An internal DNA control added at the beginning of the extraction allows the calculation of extraction and purification efficiencies.
Larger volumes of lysis buffer mix coupled with backflush increases significantly the extraction efficiency of Sterivex cartridge. A robust PCR mix supplemented with protein-based additives improved the qPCR reaction for inhibitor-rich environmental samples, but purification by PowerClean Kit prior to quantification may be still essential for some inhibitor-rich samples from coastal area. The qPCR assay condition could be specific to our sampling sites and further studies are required to investigate the practicality in other areas where different types of inhibitors exist.
This protocol improvises monitoring tools in various levels of the workflow, and thus increases the reliability of the eDNA data to infer the dynamics of environments in quantitative and qualitative perspectives. A boost in extraction yield by to folds could deepen the population or community coverage significantly, allowing the detection of rare organisms which could be easily missed due to low extraction yield. Fertilized eggs of chum salmon [ Oncorhynchus keta Walbaum, ] were obtained from Unosumai hatchery, Iwate, Japan.
The eggs were fertilized artificially on 4th December and eye-stage embryos were transferred to the Atmosphere and Ocean Research Institute, Chiba, Japan. Hatched salmon fries were fed with commercial diet after emergence in freshwater. Salmon juveniles were transferred to seawater tanks after 2-months culture in freshwater. Twenty-five individuals fork-length: ca.
Two concentrations of salmon water were prepared. To standardize filtration conditions, 1 L of salmon water at either concentration was filtered through a Sterivex cartridge 0. After filtration, the outlet of the Sterivex cartridge was sealed by Parafilm and 1. All the Sterivex cartridges were then randomly assigned to different extraction protocols after the filtration step to reduce bias on filtration order.
Representative photos of Sterivex cartridges with different extraction protocols. A A cartridge extracted by 0. Note the water mark that is present on the filter surface white arrow , suggesting insufficient contact with the lysis buffer mix. B A cartridge extracted by 2. Note that no water mark is found as in A. C A cartridge centrifuged by angled rotor. Note that remaining lysate is present black arrow.
All the connectors, stoppers, and silicon tubes were decontaminated by bleaching before use 5. The RNAlater in the Sterivex cartridges was thawed on ice and removed by aspiration through the outlet.
A silicon tube 1. We tested three protocols that were modified from a published method that was widely used in the eDNA field Buffer AL and proteinase K purchased from the same sources when necessary.
The major modifications are summarized in Fig. For Protocol 1, we followed the original extraction protocol The rotation was omitted because the filter surface was completely covered by the lysis buffer mix.
After the incubation, each Sterivex cartridge was placed in a spin column maxi spin, flat bottom, Ciro Manufacturing Corporation, Deerfield Beach, FL, USA attached to a 50 mL centrifuge tube, with the cartridge inlet facing downward.
The cartridge and spin column were removed and molecular grade ethanol The mixture was loaded on a DNeasy Blood and Tissue Kit column attached to a vacuum manifold and the column was washed by 0. The DNeasy column was dried by centrifugation at 17, g for 2 min. Six replicates of Sterivex cartridges were used for each protocol. To test the extraction efficiency, each Sterivex cartridge was extracted 3 times and the eluted samples were analyzed separately. To test the binding capacity of the DNeasy column, we performed an additional experiment to determine whether the quantity of sample input will have a saturating effect on the column binding.
The extraction was performed using Protocol 3. Since we hypothesized that the binding capacity of one DNeasy column could be saturated by high input, we compared the eDNA extracted by a single column or double columns connected in series. In the case of double columns, the elution was performed individually for the upper and lower columns, and the eluates were analyzed separately.
Negative control was prepared by filtering distilled water 1 L. The water samples were filtered and treated with RNAlater as described in previous section. The cartridges were extracted according to the Protocol 3 described in previous section. The extraction efficiency was calculated from ratio of control plasmid quantified from the DNeasy-extracted samples relative to input quantity.
To estimate the PCR inhibitor effects from samples without purification by PowerClean Pro Cleanup Kit, an additional assay was performed with 10 5 copies of chum salmon standard spiked into each PCR reaction. The chum salmon standard used in spiking is the same plasmid DNA 5 used for constructing the standard curve. A reduction in spiked values calculated by subtracting the spiked copy number to original copy number indicates that PCR reaction was not optimal.
Real-time PCR was performed on the serially-diluted inhibitor-rich sample and the slope of the dilution curve was determined. A pooled eDNA sample was made from mixing some inhibitor-rich samples from the 34 random environmental samples, and subsequently used in this preliminary test. The PCR amplification plots were compared between chum salmon standard and the pooled sample. Slope of the PCR amplification plot was obtained by linear regression on the steepest linear portion of the curve.
PCR reaction was compromised by the inhibitor when we observed a reduction in saturated fluorescence and a flatten slope of the linear portion of the amplification plot of the pooled sample in comparison to those of standard. We considered that the rescue from reduction in saturated fluorescence and slope change are indicating an increase in resistance to PCR inhibitors by the additive.
Purification efficiency of PowerClean Pro Cleanup Kit was calculated from paired samples quantified before and after the purification. Cristescu, M.
Uses and misuses of environmental DNA in biodiversity science and conservation. Article Google Scholar. Ushio, M. Demonstration of the potential of environmental DNA as a tool for the detection of avian species. Yatsuyanagi, T. Environmental DNA monitoring for short-term reproductive migration of endemic anadromous species, Shishamo smelt Spirinchus lanceolatus.
DNA 00 , 1—10 The 'old' and fresh were the same, so I don't think it was bad. Your explanation confirmed what I thought may have been happening. The region is very GC rich, that is why I am using the enhancers. I guess they help when my denature temp is low, but once I raised the temp, then all the enhancers weren't helping.
I guess Mg may be the way to go. Hi, do you mean to add an additional 4mM? Thanks Sorry, i should have said up to 4 mM MgCl2 an additional 2. So add 2. Therefore, additional factors must exist which contribute to the reduced PCR performance resulting in a high variability and failed amplification of larger fragments. This failure cannot be explained by the presence of DNA-protein and DNA-DNA crosslinks, which are reported to be mainly reversible by hydrolysis especially at elevated temperatures [ 32 — 34 ].
It is shown that this inhibition affects larger amplicons to a higher extent which leads to a complete failure of amplification of fragments above a certain size. Multiple factors are likely to play a role and further studies are needed to address this new question. The fragmentation process not only affects the target sequence and leads to an absence of template DNA, but also leads to randomly generated short DNA debris.
This debris might act directly as an inhibitor of the DNA polymerase. As an enzyme inhibitor the DNA debris might bind to the polymerase and decrease its activity resulting in a decreased reaction speed. Accordingly, the inhibition can be alleviated by increasing the substrate concentration dNTPs or the enzyme concentration polymerase. In addition, the prolongation of the reaction time elongation and extension step during PCR would allow the complete bio-polymerization of the DNA strand even at a lowered reaction speed.
This is in concordance with the findings from this study that an increase of dNTP and polymerase concentration in addition to a prolonged PCR cycling helped to reduce inhibition.
Reversible inhibitors bind non-covalently resulting in different types of inhibition depending on whether these inhibitors bind to the enzyme, the enzyme-substrate complex, or both. Four kinds of reversible enzyme inhibitions exist. During uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex while during mixed inhibition, the inhibitor can bind to the enzyme as well as its substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice versa.
Non-competitive inhibition is a kind of mixed inhibition where the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. During competitive inhibition, the substrate is unable to bind to the enzyme. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate would bind. Accordingly, the substrate and inhibitor compete for access to the enzyme's active site.
Such competitive inhibitors are often similar in structure to the substrate. The latter type of inhibition is likely to be mainly responsible for the observed phenomenon of PCR inhibition.
Clearly, more detailed kinetic studies to elucidate the type of inhibition are needed. Another mechanism which might be considered responsible for PCR inhibition could be a reduced processivity leading to a failed amplification of larger amplicons.
The DNA debris might hybridize to the template DNA, thus impairing the polymerization and leading to a dissociation of the polymerase-template complex. Such exonuclease activity might help the Taq polymerase to remove DNA fragments which hybridize unspecifically to the template strand.
A reduced processivity might then be balanced by an increase of the reaction speed due to an increase of dNTP and polymerase concentration. In summary, this study showed that polymerases from two fundamentally different sources from the archaeum Pyrococcus furiosus and the bacterium Thermus aquaticus are affected by PCR inhibition.
Therefore, these findings seem to be valid for DNA polymerases in general. Finally, this study presents solutions to overcome this newly described PCR inhibition which will allow for valuable improvements in all areas of nucleic-acid based molecular biological research and diagnostics.
Especially, in clinical routine diagnostics where the highly accurate diagnosis for a single patient is mandatory, template DNA is in some cases not limited and the costs for additional polymerase are negligible. Analyzed the data: DD BU. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Formalin-fixed and paraffin-embedded FFPE tissues represent a valuable source for biomarker studies and clinical routine diagnostics.
Funding: No current external funding sources for this study. Introduction Formalin-fixed, paraffin-embedded FFPE tissue is the most commonly used source for tissue based molecular biological testing.
Bisulfite Treatment The bisulfite conversion and subsequent purification was performed as previously described [ 19 ] with minor modifications. Download: PPT. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. References 1.
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