Implementation of a T4 Extraction Control for Molecular Assays of Cerebrospinal Fluid and Stool Specimens

Abstract

The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Extraction controls and checks for inhibitors of amplification in cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. Other specimen types , such as stool, frequently contain amplification inhibitors. Failure to test for these inhibitors can result in the reporting of false-negative results. The goal of this study was to evaluate the use of a T4 bacteriophage as an extraction and amplification control for acellular specimens. The T4 bacteriophage assay was evaluated for use as a control in 290 specimens , including cerebrospinal fluid , serum , and filtered stool. The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Inhibitors present in some patient specimens decrease the efficiency of amplification and, if not controlled, can result in the reporting of false-negative results. False-negatives may also arise from undetected extraction failures. Accrediting agencies expect diagnostic laboratories to control for these variables. To meet quality assurance standards recommended by accrediting agencies, a method to detect the presence of inhibitors, while simultaneously providing a control for extraction in acellular specimens, is needed. Extraction controls and amplification inhibitor checks for cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. In our experience, genomic DNA targets are successfully amplified in only approximately 40% of cerebrospinal fluid (CSF) specimens. Other acellular specimen types, such as stool, frequently contain amplification inhibitors.Several forms of internal controls have been evaluated, including plasmid DNA, ribosomal RNA, armored RNA, and Lambda phage. Plasmid-derived DNA may be degraded before extraction because it is unprotected and subjected to nucleases. 1 To protect the plasmid DNA, further packaging is required in a bacteriophage, which adds more time and expense to the control. Ribosomal 16 or 18S RNA is a convenient control for reverse transcription-polymerase chain reaction (PCR). Armored RNA is a nucleic acid packaged in bacteriophage coat proteins that protect the RNA from ribonucleases. The RNA is released from its protective coat during the extraction procedure and can be used as a control for reverse transcription-PCR. 2 The disadvantage of this technology is that it is proprietary and expensive, and the reverse transcription process adds unnecessary time and expense to DNA-targeted assays. Additionally, inhibitors of reverse transcription and of PCR cannot be distinguished. Specially engineered Lambda phage DNA fr...

RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests

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Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

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