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.

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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 C_T values and ensures longer storage of standards without degradation (see figure Reliable dilution and storage of RNA standards).

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).

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).

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.