Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis

Abstract

Background: Several viruses can cause diarrheal disease, a leading cause of morbidity and mortality worldwide. Existing diagnostic methods include ELISA and nucleic acid amplification, usually performed individually.

Objectives: (1) To develop a multiplexed assay for simultaneous detection of major enteric viral pathogens. (2) Quantitation of viral load by normalizing with an extrinsic control.

Study design: A simple protocol combining a one-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) with microsphere-based fluorescence detection was developed for norovirus GI and GII, rotavirus, astrovirus, sapovirus, and adenovirus. An extrinsic control, bacteriophage MS2, was spiked into each fecal sample before nucleic acid extraction to normalize between samples for the efficiency of nucleic acid extraction and amplification.

Results: The fluorescent results were quantitative and nearly as sensitive as the corresponding singleplex real time RT-PCR (qRT-PCR) assay on analytic samples. Upon testing 229 fecal samples from inpatients with diarrhea in Tanzania the assay yielded between 88% and 100% sensitivity and specificity for all analytes. The difference in fluorescence intensities of MS2 between samples indicated variable extraction efficiency and was used to better refine the viral load of each specimen.

Conclusions: This one-step nucleic acid-based assay enables rapid, sensitive and specific detection of the major viral causes of gastroenteritis. The quantitation yielded by the assay is informative for clinical research particularly in the context of mixed infections.

Figure 1

Correlation between template copy number, detection by singleplex qRT-PCR (Ct), and multiplex RT-PCR-Luminex assay (median fluorescence intensity – negative control). A serial dilution of T7 RNA transcript for were prepared either alone in 1 mM sodium citrate (buffer) or spiked into a common fecal nucleic acid extract. Materials were then interrogated by both singleplex qRT-PCR and the multiplex RT-PCR-Luminex assay. Best fit lines and regression (R²) were extrapolated. The rest of the analytes are presented in supplemental Figure S1.

Figure 2

Correlation between singleplex qRT-PCR results and the multiplex RT-PCR-Luminex assay on clinical specimens. Two hundred twenty nine diarrheal specimens from inpatients in Tanzania underwent nucleic acid extraction and testing by both singleplex qRT-PCR and the multiplex RT-PCR-Luminex assay. Specifically, these included 29 qPCR positive specimens for rotavirus, 20 for norovirus GII, 5 for adenovirus, 3 for sapovirus, 2 for norovirus GI and 1 for astrovirus. Cut-off threshold values were determined by ROC analysis (see Table 2).

Figure 3

Receiver-Operating Characteristic (ROC) analysis for the multiplex RT-PCR-Luminex assay versus singleplex qRT-PCR as the gold standard.

Figure 4

Correlation between singleplex qRT-PCR Ct and the multiplex RT-PCR-Luminex assay MFI values on clinical specimens. Best fit lines and regression (R²) were extrapolated.

Figure 5

Quantitation of viral load and normalization for the sample’s extraction and amplification efficiency. The viral load was estimated from the multiplex RT-PCR-Luminex positive samples based on the standard curves of Figure 1 and S1, then multiplied by the assay dilution factor to yield viral copies per gram of stool. This viral load was then adjusted for the sample’s nucleic acid extraction and RT-PCR amplification efficiency. Specifically, for each sample the loss of detection of the extrinsic control was estimated by comparing the expected amount of spiked MS2 vs. the detected amount of MS2. Three cases of co-infection were included and shown in color, i.e. one norovirus GI and GII coinfection in green, one norovirus GII and rotavirus coinfection in red, one norovirus GII and sapovirus coinfection in blue.

Figure 6

Simplified estimation for viral load. The ratio of viral ΔMFI value to extrinsic control ΔMFI (ΔMFI_Analyte/ΔMFI_{ExtrinsicControl}) is shown versus the adjusted viral load values of Figure 5. Norovirus GII and rotavirus shown.