Evaluation Of Pre-analytical Variables And Biohazard Exposure Risk In The Detection Of Influenza Virus By Nucleic Acid-based Assay

Background PCR-based nucleic acid (NA) testing is increasingly the first-choice method for virological surveillence and diagnosis of influenza. However, not only used NA assay protocols, reagents, primers and probes, but also pre-analytical variables, including the storage and transport of samples and NA purification methods, have major effects on the performance of NA assay. And, potential biohazard exposure to personnel during NA assay must be taken into consideration and would affect practical use of NA assay.Objective1) To apply Maxwell 16 System, an automated NA extraction platform based-on magnetic bead technology, to automated extraction of influenza virus (flu-v) RNA for diagnosis of flu using PCR-based testing and evaluated its performance and suitability for detection of flu-v from various samples.2) To assess the effects of lysis buffers of different NA extraction methods on the stability of flu-v RNA in order to lay a foundation for the utilization of lysis buffer as preservative of flu-v samples.3) To evaluate the biohazard risk during extraction of flu-v RNA by examining the ability of lysis buffers of frequently used NA extraction methods to inactivate flu-v.Methods1) Following extraction by Maxwell 16 System and QIAamp Viral RNA Mini Kit (QIAamp Kit) from flu-v stock dilution series, throat swabs, bronchoalveolar lavage fluid (BALF) and fecal samples of human and poultry origin, the extracted RNA was assayed by the CDC-developed real-time RT-PCR (rRT-PCR) protocols. The performance of Maxwell 16 System, including sensitivity, linearity, precision and extraction efficiency of flu-v RNA from the various clinical and field samples, was evaluated by comparison with QIAamp Kit based on silica gel column chromatography, a common used method for extraction of flu-v RNA.2) Influenza virus stocks were treated respectively with two lysis buffers, Lysis Solution (Lysis Buffer+Proteinase K) from Maxwell 16 System and Buffer AVL with carrier RNA from QIAamp Kit, and the lysis buffer-virus mixtures were kept at 22℃,4℃,—20℃or underwent cyclic freeze-thaw. The relative quantity and quality of viral RNA after storage or freeze-thaw were evaluated with an rRT-PCR assay and a conventional RT-PCR assay targeting full-length matrix gene of flu-v.3) Three lysis buffers, Lysis Solution, Buffer AVL and RNA Extraction Reagent A (ERA) from a modified guanidinium isothiocyanate kit, which based on three different methods commonly used in viral RNA extraction, were selected to process high-titer flu-v stocks (105.7TCID50/ml) according to manufacturer's instructions and preliminary research of ours. The lysis buffer-virus combinations were then serially tenfold diluted using virus growth medium and the dilutions were inoculated onto MDCK cells for virus isolation. Cell growth was observed daily under an inverted microscope. Cultures were harvested and hemagglutination tests were done with the supernatant when cytopathic effect (CPE) of cultured cells developed more than 75%.Results1) Extraction with Maxwell 16 System standard procedure (Maxwell 16-S) resulted in good linearity across a wide range, low inter-and intra-run variation and higher analysis sensitivity compared to those with the QIAamp extraction. Maxwell 16-S extraction yielded more RNA and/or fewer PCR inhibitors from throat swabs and BALF samples than QIAamp extraction but they had opposite performance for extraction from fecal samples. When extraction with a modified procedure (with reduced input and increased output) of Maxwell 16 System (Maxwell 16-M), the improved capacity to remove PCR inhibitors contributed to an enhanced sensitivity for detection of flu-v in poultry fecal samples although still a relative low sensitivity in human fecal samples compared to those with QIAamp Kit.2) Entire matrix gene was amplified from samples treated with either of the lysis buffers after storage of 160 days at -20℃or five times of freeze-thaw, and the relative intensity of amplification products did not show to decline. Viral RNA in Buffer AVL degraded more rapidly than in Lysis Solution when kept at 22℃and 4℃, while the integrity of viral RNA could be maintain for certain time intervals at 4℃in both of the lysis buffers.3) Massive cell death occurred within 24 hours after inoculation of lysis buffer-virus mixtures at low dilutions as well as the corresponding reagent controls at low dilutions. Cytopathic effect did not develop even after three blind passages of all the lysis buffer-virus mixtures at high dilutions and no viable virus in the cultures was detected by the hemagglutination tests, while infectious virus was isolated in the virus controls diluted at the same concentrations.Conclusion1) Qualitative and quantitative detection of flu-v is feasible using Maxwell 16 System in combination with rRT-PCR. But it differs in capacities of removing PCR inhibitors from different sample matrices, thereby having impacts on the sensitivity of rRT-PCR assay. A properly reduction of input volume of sample extracted with Maxwell 16 System for challenging matrices may improve detection.2) The Maxwell 16-S procedure is suitable for extraction of flu-v RNA from throat swab and BALF samples, and the Maxwell 16-M procedure is suitable for extraction from poultry fecal samples, while a more optimized Maxwell 16 procedure is needed to develop for extraction from human fecal matrix.3) Intact and non-decreased influenza virus RNA could obtained from samples preserved in lysis buffer underwent cyclic freeze-thaw or a long period of storage at refrigeration and frozen temperatures. However, lysis buffers different in methodology have not always the same ability to stabilize viral RNA.4) The lysis buffers of the three nucleic acid extraction methods could render high-titer influenza virus non-infectious according to the procedures recommended by the manufacturers. Thus the risk of biohazard exposure during NA extraction was significantly reduced after initial lysis of flu-v samples, which has implications for moving processing of flu-v samples within an automated extractor outside of a biosafety cabinet or under low containment laboratories, and for application of lysis buffer as stabilizing agent of flu-v samples during storage and transport.