PCR Applications — Pathogen Detection

Rapid, sensitive, and highly specific PCR-based detection of pathogen nucleic acids
Molecular methods for detecting pathogens are becoming increasingly popular, as they offer accurate detection at a fraction of the time and effort invested in traditional, culture-based methods. In particular, both real-time and end-point PCR deliver rapid, sensitive, and highly specific detection of nucleic acids from bacteria, viruses, fungi, and other microbial organisms.
Real-time PCR-based pathogen detection
End-point, one-step RT-PCR-based virus research
The importance of internal controls in real-time PCR-based pathogen detection
Manufacturing according to Good Manufacturing Practice (GMP) principles
Real-time PCR-based pathogen detection
When applying real-time PCR and RT-PCR to the research of pathogen nucleic acids (DNA and/or RNA), the inclusion of an internal, positive control is often desired to rule out the possibility of false negatives. In other words, a multiplex reaction is carried out to quantify both the target nucleic acids, as well as control nucleic acid. Performing multiplex PCR and RT-PCR also provides the advantage of detecting several pathogens from the same sample simultaneously, which saves times and conserves sample. The QuantiFast Pathogen +IC Kit includes the QIAGEN Internal Control and delivers sensitive detection of low target amounts. It is suitable for analysis of up to 4 RNA and/or DNA templates plus the internal control (see figures High linearity and precision of singleplex and duplex detection, Sensitive detection of Norovirus on the Rotor-Gene Q, and Sensitive detection of BHV-1 on the ABI 7500). QuantiTect Virus Kits, which are specially designed for sensitive detection of viral DNA and/or RNA, use similar multiplex PCR technology to QuantiTect Multiplex Kits, allowing detection of up to 4 targets in a single reaction.  As little as 1 copy of viral DNA or RNA target can be detected either in multiplex reactions with an internal control or in single-target amplification reactions (see figure Improved detection of low amounts of viral RNA).

Multiplex assays without optimization are enabled by QuantiFast Pathogen or QuantiTect Virus PCR Buffers, which contain an optimized combination of KCl and (NH4)2SO4, as well as synthetic Factor MP, for effective primer annealing, and HotStarTaq Plus DNA Polymerase for a stringent hot start with a short activation time (see figure Unique PCR buffer promotes stable and efficient annealing).

QuantiTect Nucleic Acid Dilution Buffer, supplied with both kits, stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The buffer enables reliable dilution of standards used to quantify pathogen nucleic acids, giving a wide linear range, from low to high CT values and ensures longer storage of standards without degradation (see figure Reliable dilution and storage of RNA standards).

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End-point, one-step RT-PCR-based virus research
RNA secondary structure can affect RT-PCR results in a number of ways. During reverse transcription, regions of RNA with complex secondary structure can cause the reverse transcriptase to stop or dissociate from the RNA template. Truncated cDNAs that do not include the downstream primer-binding site are not amplified during PCR. In some cases, the reverse transcriptase skips looped structures, resulting in deletions in the cDNA that lead to truncated PCR products.

When amplifying a low-abundance transcript or viral sequence, these problems are even more critical. A well-balanced system, consisting of reverse transcriptase, a stringent hot-start enzyme, and an optimized buffer system is crucial for applications such as viral detection or gene expression analysis, where maximum sensitivity is often required.

The buffer provided with the QIAGEN OneStep RT-PCR Kit allows reverse transcription to be performed at an elevated temperature (50˚C; see figures Influence of annealing temperature on one-step RT-PCR specificity and QIAGEN PCR buffers increase specific primer annealing). This high reaction temperature improves the efficiency of the reverse transcriptase reaction by disrupting secondary structures and is particularly important for one-step RT-PCR performed with limiting template amounts (see figure Effective detection of viral RNA).

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The importance of internal controls in real-time PCR-based pathogen detection
PCR-based pathogen detection requires the use of appropriate controls, which aid in result interpretation by identifying adverse factors such as contamination, inhibition of the amplification reaction, or problems during nucleic acid extraction (see figure Correct interpretation of negative results). For example, ruling out the possibility that your reaction has been contaminated, leading to a false-positive result requires the use of adequate negative controls. Alternatively, ensuring that your test would have detected the pathogen, had it been present in the sample, (i.e., reducing false-negative results) requires the use of appropriate positive controls. In the context of process safety and workflow simplification, exogenous heterologous internal controls (IC) are the most informative and flexible. The amount of IC template spiked into a sample is defined and consistent, and unrestricted design options enable optimization of IC properties. Only heterologous ICs allow for a design and setup that prevents competition for PCR components, and heterologous ICs are suited as universal controls, thereby making their implementation in new assays easy.

QIAGEN has created a robust set of internal controls, which are included in the QuantiFast Pathogen PCR +IC Kit, and allow accurate detection of PCR inhibition and failure of extraction. These exogenous heterologous internal controls carry a unique artificial sequence that is not present in any biological sample material, and is detected with a dual-labeled probe assay using a MAX labeled probe (detectable in the same channel as HEX, JOE or VIC), which differs from those specific to the target pathogen. The IC DNA or RNA template is spiked into the sample during PCR or RT-PCR setup to ensure that the amplification process proceeded without problems. The IC template can also be spiked into the sample prior to the extraction process to additionally control for extraction failure. In both cases, the Internal Control RNA template additionally controls for successful reverse transcription (see figure QIAGEN Internal Control).

Because the QIAGEN Internal Control does not occur naturally in the test specimen, but is rather added to the sample, the amount of internal control template is defined and consistent, independent of sample type or sampling technique.

The internal control is detected with primers and probes distinct from those used for the target pathogen, thereby preventing primer competition during the amplification reaction. Additionally, the non-competitive design of the IC assay prevents competition for other reaction components and assay preoptimization with QuantiFast Pathogen Master Mix ensures that the sensitivity of pathogen detection is not impaired by duplexing with the IC (see figure No loss of sensitivity when including the Internal Control).

QIAGEN Internal Controls behave predictably in the presence of PCR inhibitors. A shift in the IC signal or failure of IC amplification give a clear indication of PCR inhibition or other errors, allowing the correct interpretation of negative results (see figure Correct interpretation of negative results).

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Manufacturing according to Good Manufacturing Practice (GMP) principles
The manufacturing process, quality control, and the documentation of QIAGEN´s PCR enzymes and buffers are performed using Good Manufacturing Practice principles — thereby ensuring a constantly high-quality, standardized product for all of our customers, including those within the applied testing, pharmaceutical, and diagnostic industries. In brief, our GMP system for these products encompasses the following aspects:
  • QIAGEN has been certified under DIN ISO 9001 and ISO13485 quality standards since 1998.
  • We have convincingly demonstrated the capability of consistently manufacturing products with the required quality and of complying with their required specifications.
  • Raw materials used at QIAGEN meet the highest quality standards. Each supplier is managed according to QIAGEN´s strict documented qualification program.
  • Our manufacturing processes are clearly defined and systematically reviewed. Manufacturing, as well as testing documentation is under strict change control.
  • Instructions and procedures are written in clear language using Good Documentation Practices (GDP). Processes are documented via a sophisticated computerized document management system. Operators are trained to carry-out and document procedures.
  • QIAGEN completely tracks products from raw material to customer. Records of manufacture (including distribution) demonstrate that all steps required by the defined procedures and instructions were taken, and that the quantity and quality of the product was as expected. These records enable the complete history of a batch to be traced and are retained in a comprehensible and accessible form.
  • Manufacturing processes take place under clean room conditions (class 100.000) in an access controlled area. The critical process steps are performed under laminar flow class 100. The relevant production steps and associated clean rooms are continuously monitored for bioburden particle, temperature, and moisture. Our facilities are designed to a defined flow of personnel and material.
  • A highly optimized purification process is used for the DNA polymerases and RT enzymes, enabling an extremely low level of residual DNA content (including host) that allows amplification of a wider range of targets, including Escherichia coli. All equipment that has direct contact with the product is sanitized or is disposable. All equipment is qualified to fulfill the process requirements and is dedicated wherever possible for a single product.

 


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High linearity and precision of singleplex and duplex detection.
High linearity and precision of singleplex and duplex detection.
A 6-log range of both Norovirus RNA singleplex detection and Norovirus/IC duplex detection shows high precision and linearity. Error bars each represent ±1 SD of 3 real-time RT-PCR replicates.
Improved detection of low amounts of viral RNA compared with other real-time kits.
Improved detection of low amounts of viral RNA.
Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit or kits from Suppliers AII and I. The QuantiTect Virus Kit provided much higher sensitivity than the other reagents tested, enabling reliable analysis of unknown samples. ND: Not detected after 50 PCR cycles.
ID_0259_QFPathogen
Sensitive detection of Norovirus on the Rotor-Gene Q.
A Norovirus RNA transcript was serially diluted (100 to 10-5) and detected by either singleplex real-time RT-PCR or by duplex real-time PCR in parallel with the QIAGEN IC. Real-time PCR was carried out using the QuantiFast Pathogen RT-PCR +IC Kit on the Rotor-Gene Q without any PCR optimization. Duplex reactions contained a fixed amount of IC template. Each dilution was analyzed in triplicate; one replicate per dilution is shown.
Sensitive detection of BHV-1 on the ABI 7500
Sensitive detection of BHV-1 on the ABI 7500.
Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the internal control (IC). Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the ABI 7500 according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of IC template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.
Unique Type-it Microsatellite PCR Buffer Promotes Stable and Efficient Annealing
Unique PCR buffer promotes stable and efficient annealing.
[A] NH4+ ions prevent nonspecific primers from annealing to the template. Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. [B] Together with K+ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.
Reliable dilution and storage of RNA standards using QuantiTect Nucleic Acid Dilution Buffer.
Reliable dilution and storage of RNA standards.
Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at –20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower CT values and improved stability of the standards.
Influence of Annealing Temperature on One-Step RT-PCR Specificity
Influence of annealing temperature on one-step RT-PCR specificity.
One-step RT-PCR was performed using kits from the indicated suppliers over a range of annealing temperatures. A 1289 bp fragment from the human RCC1 gene was reverse transcribed and amplified from Hela RNA (arrow). M: markers. High levels of specific amplification without optimization were observed only with the QIAGEN OneStep RT-PCR Kit.
Effective detection of viral RNA
Effective detection of viral RNA.
A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplified from Sendai virus RNA isolated from persistently infected Vero cells. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit and the indicated number of viral genome copies. M: markers.
Data kindly provided by H. Rausch, Max Planck Institute for Biochemistry, Martinsried, Germany as part of the project “Experimental control of virological work at safety levels 2 and 3 in Bavaria,” supported by the Bavarian Ministry of the Environment.
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
QIAGEN PCR buffer increases specific primer annealing.
K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer–template binding over a wide temperature range.
Correct interpretation of negative results.
Correct interpretation of negative results.
Duplicates of two concentrations of a viral RNA target were co-amplified with the QIAGEN IC in the presence of different amounts of a PCR inhibitory substance (humic acid) on the Rotor-Gene Q. [A] No template controls (NTCs) serve as a reference for IC signal. [B] Amplification of viral RNA target and IC confirms successful amplification. [C] The IC indicates the presence of a low amount of inhibitors. [D] Failure to detect the IC shows the failure of the amplification reaction through presence of inhibitors.
QIAGEN Internal Control workflow
QIAGEN Internal Control workflow.
A control to monitor the extraction and/or reverse transcription and amplification steps. The QIAGEN Internal Control can be added during purification to act as an extraction and amplification control, or during PCR analysis to act as an amplification control.
No loss of sensitivity when including Internal Control
No loss of sensitivity when including Internal Control.
Singleplex detection of Influenza A RNA from vaccine using the QuanitTect Virus Kit or a kit from Supplier I was compared with duplex detection with the QuantiFast Pathogen RT-PCR +IC Kit. The Influenza A RNA was subjected to a 1:4 dilution series and an Agilent (Stratagene) Mx3005p cycler was used. The same reliable performance was achieved with QuantiFast Pathogen RT-PCR +IC Kit as with the QuantiTect Virus Kit and higher sensitivity was achieved compared with the kit from Supplier I.