There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20–200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China.
Viral respiratory tract infections are a common cause of hospitalization and mortality in infants and young children. Influenza A, influenza B, parainfluenza, human rhinovirus, adenovirus, respiratory syncytial virus, metapneumovirus, and coronavirus are recognized as viral agents associated with respiratory tract infections [
Existing standard nonmolecular diagnostic methods such as viral culture and immunofluorescence (DFA) are time-consuming, labor intensive, or have low sensitivity [
In our previous study, a multiplex PCR based on the GenomeLab Gene Expression Profiler (GeXP) analyzer had been shown to be effective for the detection of pandemic influenza A H1N1 virus [
A total of 247 nasopharyngeal aspirates were collected in the Children’s Hospital of Beijing, China, during June 2008 and March 2010 from hospitalized children under two years of age presenting acute lower respiratory infection (ALRI) syndromes based on clinical measurements recommended by the World Health Organization (WHO). The patients were included in this study according to the following criteria: parenchymal infiltration on chest radiography, dry or moist rale, body temperature above 37.5°C, a normal or low leukocyte count, or lower chest wall indrawing. The mean age of study participants was 13.08 months. Of 247 cases, 149 (60.32%) participants were male and 98 (39.68%) female. A total of 160 (64.78%) participants were from urban areas and 87 (35.22%) were from suburban or rural areas of China (Hebei, Henan, Shanxi, Shandong, and Inner Mongolia). The specimens were collected within 5 to 7 days after symptoms onset. Patients with bacterial infections were excluded. All aspects of the study were performed in accordance with national ethics regulations and approved by the Institutional Review Boards of the Center for Disease Control and Prevention of China. Children's parents were apprised of the study’s purpose and of their right to keep information confidential. Written consent was obtained from children's parents.
Total RNA/DNA was extracted from 200
Nine pairs of chimeric primers [
Primer information.
Primer | Sequence |
Gene | Size (bp) | Concentrationsa |
---|---|---|---|---|
FluA F |
|
M | 270 | 75 nM/L |
FluA R |
| |||
FLuB F |
|
M | 166 | 50 nM/L |
FLuB R |
| |||
sH1N1 F |
|
HA | 250 | 75 nM/L |
sH1N1 R |
| |||
PIV1 F |
|
HA | 284 | 87.5 nM/L |
PIV1 R |
| |||
PIV2 F |
|
HA | 194 | 50 nM/L |
PIV2 R |
| |||
PIV3 F |
|
HA | 230 | 75 nM/L |
PIV3 R |
| |||
HRV F |
|
|
144 | 50 nM/L |
HRV R |
| |||
HMPV F1 |
|
L | 208 | 100 nM/L |
HMPV R1 |
| |||
HMPV F2 |
|
N | 208 | 50 nM/L |
HMPV R2 |
| |||
Adv F |
|
Hexon | 338 | 100 nM/L |
Adv R |
| |||
NL63 F |
|
Polym-erase | 176 | 50 nM/L |
NL63 R |
| |||
OC43 F |
|
N | 200 | 50 nM/L |
OC43 R |
| |||
229E F |
|
N | 183 | 50 nM/L |
229E R |
| |||
HKU1 F |
|
N | 220 | 50 nM/L |
HKU1 R |
| |||
RSVA F |
|
F | 158 | 50 nM/L |
RSVA R |
| |||
RSVB F |
|
F | 279 | 100 nM/L |
RSVB R |
| |||
HBoV F |
|
NP1 | 290 | 100 nM/L |
HBoV R |
| |||
Rnasep F |
|
RNase-P | 125 | 25 nM/L |
Rnasep R |
| |||
Tag F | AGGTGACACTATAGAATA | 50 nM/L | ||
Tag R | GTACGACTCACTATAGGGA |
One-Step RT-PCR Kit (Qiagen, Hilden, Germany) was used for the amplification. A total of 25
The specificity of the two-tube assay on all viral targets was tested individually in the two multiplex reactions under the experimental condition as described previously. Cell-cultured virus stocks contained in NATtrol Respiratory Validation Panels 2 (NATRVP-2) (ZeptoMetrix, Buffalo, New York, USA) were used as the positive controls of FluA, FluB, sH1N1, PIV1-3, HRV, Adv, RSVA, RSVB, HMPV, CoV 229E, and CoV OC43 to validate the specificity. Specimens genotyped previously [
To determine the analytical sensitivity of the assay, serial dilutions of viral RNA transcripts of all RNA viruses and serial dilutions of the plasmids containing the Adv and HBoV target sequence were analyzed by the two-tube assay. The PCR products were cloned into a pGEM-T vector, which was used to transform DH10B cells. The plasmid DNA was extracted with an E.Z.N.A. Plasmid Mini Kit I (Omega, GA, USA). The RNA copy number was calculated after measuring the concentration of the RNA transcribed
The Luminex xTAG RVP Fast kit (Abbott, Illinois, USA) enables users to detect simultaneously FluA, FluB, RSV, PIV1-4, Adv, HMPV, CoV 229E, NL63, OC43, HKU1, enterovirus (HEV)/HRV, and HBoV. The RNA/DNA extracted from 247 clinical specimens was tested using the Luminex xTAG RVP Fast assay in a 96-well plate format according to the manufacturer’s instructions [
The additional targets detected only by the two-tube assay were confirmed by independent PCR and sequencing. The sequencing was performed using T7 and SP6 sequence primers on AB SOLiDTM 4.0 System (Applied Biosystems, USA) and compared with the sequences in GenBank for pathogen identification by using the BLAST algorithm. The additional targets detected only by the Luminex xTAG RVP Fast assay were resolved using remaining extracts with the Seeplex RV15 ACE detection kit (Seegene, Seoul, Korea) [
All statistical analyses were performed using Statistical Package for Social Sciences (SPSS) software (version 13.0) for Windows. The
The expected size of each virus type/subtype-specific amplicon was observed and separated clearly from the other viral targets on QIAxcel automatic electrophoresis for all of the positive controls. No mispriming (primer dimer) or other amplification in the negative controls was observed in either tube (Figure
Specificity analyses of the the two-tube assay based on the automatic electrophoresis. All positive controls were identified successfully, and no mispriming was observed in either tube. Lanes 1 to 9 show the results of amplification of HRV (148 bp), FluB (171 bp), CoV 229E (189 bp), CoV OC43 (208 bp), CoV HKU1 (226 bp), FluA (sH1N1) (257 bp and 276 bp), FluA (H3N2) (276 bp), PIV1 (286 bp) and Adv (342 bp), respectively. Lanes 11–17 show the results of amplification of RSVA (163 bp), CoV NL63 (179 b), PIV2 (198 bp), HMPV (212 bp), PIV3 (233 bp), RSVB (280 bp), and HBoV (298 bp), respectively. For the viral targets that used clinical specimens as positive controls, including HBoV, CoV HKU1, and CoV NL63, a single PCR product was detected in addition to the internal control peak (126 bp). Lanes 10 and 18 are the negative controls (distilled water) of tube 1 and tube 2, respectively. Lane MM, molecular marker.
The sensitivity of the assay was evaluated for each virus type/subtype individually using serial tenfold dilutions ranging from 10 to 105 copies of cloned PCR products in both of the two tubes. The limit of detection for HRV, PIV2, PIV3, RSVA, HBoV, and Adv was 20 copies per reaction and 200 copies per reaction for the other 10 virus type/subtypes assayed in this study.
The sensitivity of two-tube assay when all the viral targets were present was also tested. The detection sensitivity in tube 1 with 9 pre-mixed viral targets was 2000 copies per reaction and 200 copies per reaction in tube 2 with 7 premixed templates (Figure
Sensitivity analyses of the two-tube assay based on the automatic electrophoresis. Lanes 1 to 5 contain PCR products of pre-mixed viral targets in tube 1 of 2 × 106 to 200 copies. Lanes 7 to 11 contain PCR products of pre-mixed viral targets in tube 2 of 2 × 105 to 20 copies. Lanes 6 and 12 are the negative controls of tube 1 and tube 2, respectively. Lane MM, molecular marker. The detection sensitivity in tube 1 with 9 pre-mixed viral targets was 2000 copies per reaction and 200 copies per reaction in tube 2 with 8 pre-mixed templates. Only the amplicons of sH1N1 and CoV 229E were absent with dilutions of 200 copies of pre-mixed viral targets in tube 1 (Lanes 5), and only the amplicons of CoV NL63, HMPV, and RSVB were absent with dilutions of 20 copies of pre-mixed templates in tube 2 (Lanes 11). The primer dimers which were inevitable in the multiplex PCR are increasingly apparent with the dilution of template.
All the comparative detections were double-blind tests performed by trained staff in our laboratory. All of the 247 specimens assayed with the Luminex xTAG RVP Fast assay were retested by the two-tube assay. The two assays detected presence of at least one of the 16 assayed viruses in 194 specimens, and the two assays both detected virus in 181 specimens. The two-tube assay detected virus in 8 specimens that were negative in the Luminex xTAG RVP Fast assay (2 Adv, 3 RSVB, 2 HRV, and 1 PIV3), and 5 specimens had a positive detection of HRV only by the Luminex xTAG RVP Fast assay. The Luminex xTAG RVP Fast assay identified 111 specimens to be coinfections, 83 of them (74.77%) were in complete agreement between both assays, and detection of 13 viruses (in 12 specimens) was missed by the two-tube assay. Additional 14 viruses (in 14 coinfected specimens) were detected only by the two-tube assay, and 2 coinfection specimens (no. 28 and no. 118) gave inconsistent results between the two assays (Table
As shown in Table
Performance of the two-tube assay for individual target compared with the Luminex xTAG RVP Fast assay.
Viruses | No. of specimens: two-tube/Luminex xTAG RVP Fast assay | Performance of the two-tube assay compared with the Luminex xTAG RVP Fast assay | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
+/+ | +/− | −/+ | −/− | Sensitivity% | Specificity% | PPV% | NPV% | Accordance rate% | Kappa | |
FluA | 20 | 0 | 0 | 227 | 100 | 100 | 100 | 100 | 100 | 1.000 |
sH1N1 | 14 | 0 | 2 | 231 | 87.50 | 100 | 100 | 99.14 | 99.19 | 0.929 |
FluB | 1 | 0 | 0 | 246 | 100 | 100 | 100 | 100 | 100 | 1.000 |
PIV1 | 1 | 0 | 0 | 246 | 100 | 100 | 100 | 100 | 100 | 1.000 |
PIV2 | 2 | 0 | 0 | 245 | 100 | 100 | 100 | 100 | 100 | 1.000 |
PIV3 | 22 | 2 | 0 | 223 | 100 | 99.11 | 91.67 | 100 | 99.19 | 0.952 |
HRV | 95 | 15 | 13 | 124 | 87.96 | 89.21 | 86.36 | 90.51 | 88.66 | 0.770 |
HMPV | 27 | 1 | 0 | 219 | 100 | 99.55 | 96.43 | 100 | 99.60 | 0.980 |
Adv | 24 | 11 | 1 | 211 | 96.00 | 95.05 | 68.57 | 99.53 | 95.14 | 0.773 |
CoV NL63 | 1 | 0 | 0 | 246 | 100 | 100 | 100 | 100 | 100 | 1.000 |
CoV OC43 | 11 | 0 | 0 | 236 | 100 | 100 | 100 | 100 | 100 | 1.000 |
CoV 229E | 2 | 0 | 0 | 245 | 100 | 100 | 100 | 100 | 100 | 1.000 |
CoV HKU1 | 3 | 0 | 0 | 244 | 100 | 100 | 100 | 100 | 100 | 1.000 |
RSVA | 7 | 1 | 0 | 239 | 100 | 99.58 | 87.50 | 100 | 99.60 | 0.931 |
RSVB | 86 | 10 | 0 | 151 | 100 | 93.79 | 89.58 | 100 | 95.95 | 0.913 |
HBoV | 28 | 0 | 5 | 214 | 84.85 | 100 | 100 | 97.72 | 97.98 | 0.907 |
The numbers of positive and negative specimens detected by both testing methods are shown. This table shows the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the kappa values for each target using the Luminex xTAG RVP Fast assay as the reference for comparison. All the kappa values were above 0.75, indicating that the two assays were in agreement.
The confirmed results for specimens with discordant results between the two-tube assay and the Luminex xTAG RVP Fast assay.
Case no. | Two-tube | Luminex xTAG RVP Fasta | Seeplex | Confirmed results |
---|---|---|---|---|
43 | HRV | HEV/HRV, |
HRV | HRV |
349 | HRV, RSVB | HEV/HRV, RSVB, and |
HRV, RSVB | HRV, RSVB |
352 | HRV, RSVB | HEV/HRV, RSVB, and |
HRV, RSVB | HRV, RSVB |
146 | HMPV | HMPV, |
HMPV | HMPV |
149 | HMPV | HMPV, |
HRV, HMPV |
|
16 | None |
|
HRV |
|
125 | RSVB | RSVB, |
HRV, RSVB |
|
262 | RSVB, PIV3, and HBoV | RSVB, PIV3, HBoV, and |
PIV3, RSVB | RSVB, PIV3, and HBoV |
270 | PIV3, RSVB, and HMPV | PIV3, RSVB, HMPV, and |
PIV3, RSVB, and HMPV | PIV3, RSVB, and HMPV |
142 | RSVB | RSVB, |
RSVB | RSVB |
21 | PIV3 | PIV3, |
PIV3 | PIV3 |
66 | None |
|
None | None |
85 | FluA, sH1N1, and HBoV | FluA (H1), HBoV, and |
FluA | FluA, sH1N1, and HBoV |
87 | None |
|
None | None |
91 | None |
|
None | None |
114 | None |
|
None | None |
274 | HRV, RSVB | HEV/HRV, RSVB, and |
HRV, Adv | HRV, |
28 | FluA, HBoV, and |
FluA |
NDd | FluA, |
118 | FluA, Adv, |
FluA |
FluA, RSVA, Adv, and HBOV | FluA, |
All of the 40 additional targets detected only by the two-tube assay were confirmed by sequencing as true positives. For those specimens with discordant results by the two-tube and RVP assays, the Seeplex RV15 ACE detection kit was used to resolve the discrepancy (Table
The confirmed results were defined as the results detected by at least two of the three PCR assays [
Performance of the two-tube assay for individual target compared with the confirmed results.
Viruses | No. of specimens by two-tube/confirmed results | Performance of the two-tube assay compared with confirmed results | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
+/+ | +/− | −/+ | −/− | Sensitivity |
Specificity |
PPV |
NPV |
Accordance rate% | Kappa | |
FluA | 20 | 0 | 0 | 227 | 100 | 100 | 100 | 100 | 100 | 1.000 |
sH1N1 | 14 | 0 | 2 | 231 | 87.50 | 100 | 100 | 99.14 | 99.19 | 0.929 |
FluB | 1 | 0 | 0 | 246 | 100 | 100 | 100 | 100 | 100 | 1.000 |
PIV1 | 1 | 0 | 0 | 246 | 100 | 100 | 100 | 100 | 100 | 1.000 |
PIV2 | 2 | 0 | 0 | 245 | 100 | 100 | 100 | 100 | 100 | 1.000 |
PIV3 | 24 | 0 | 0 | 223 | 100 | 100 | 100 | 100 | 100 | 1.000 |
HRV | 110 | 0 | 3 | 134 | 97.35 | 100 | 100 | 97.81 | 98.79 | 0.975 |
HMPV | 28 | 0 | 0 | 219 | 100 | 100 | 100 | 100 | 100 | 1.000 |
Adv | 35 | 0 | 1 | 211 | 97.22 | 100 | 100 | 99.53 | 99.60 | 0.984 |
CoV NL63 | 1 | 0 | 0 | 246 | 100 | 100 | 100 | 100 | 100 | 1.000 |
CoV OC43 | 11 | 0 | 0 | 236 | 100 | 100 | 100 | 100 | 100 | 1.000 |
CoV 229E | 2 | 0 | 0 | 245 | 100 | 100 | 100 | 100 | 100 | 1.000 |
CoV HKU1 | 3 | 0 | 0 | 244 | 100 | 100 | 100 | 100 | 100 | 1.000 |
RSVA | 8 | 0 | 0 | 239 | 100 | 100 | 100 | 100 | 100 | 1.000 |
RSVB | 96 | 0 | 0 | 151 | 100 | 100 | 100 | 100 | 100 | 1.000 |
HBoV | 28 | 0 | 0 | 219 | 100 | 100 | 100 | 100 | 100 | 1.000 |
This table shows the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the kappa values for each target using the confirmed results as the reference for comparison. All the specificity and PPV were 100%, all the accordance rate values were above 98.79%, and all the kappa values were above 0.75.
In this study, a novel two-tube assay based on the QIAxcel capillary electrophoresis to replace GeXP has been evaluated for the potential application in routine laboratory testing of respiratory viruses in China. Under optimized working condition, the two-tube assay revealed good specificity without nonspecific amplification using size detection system though conventional validation of specificity is usually done by probe detection, hybridization, nested PCR, and sequencing. The two-tube assay achieved a sensitivity of 20–200 copies per reaction when assayed individually for each virus type/subtype, and 2000 copies when 9 viral targets were present in tube 1 and 200 copies when 7 viral templates were present in tube 2. These results suggest the utility of the two-tube assay for the detection of multiple respiratory virus infections for laboratories not equipped to perform liquid chip-based multiplex assays.
The 247 specimens from hospitalized children showed a high prevalence of infection and co-infection with the common respiratory viral pathogens. HRV was found most frequently, followed by RSVB. The major discrepancies between the two-tube assay introduced in this study and the Luminex xTAG RVP Fast assay were found for the detection of HRV, Adv, HBoV, RSVB, and sH1N1 (kappa = 0.770, 0.773, 0.907, 0.913, and 0.929, resp.) (Table
The two-tube assay, as confirmed by the Luminex xTAG RVP Fast assay, revealed that almost 80% of the respiratory specimens tested were positive for at least one viral infection, and viral coinfections were frequent. The overall co-infection rates of the two-tube assay and the Luminex xTAG RVP Fast assay were 48.58% (120/247) and 44.94% (111/247), respectively. Compared with other similar reports [
The routine use of extensive testing by methods such as the two-tube assay should make large studies of the clinical relevance of single and multiple respiratory virus infections more feasible. The clinical relevance of the outcome of multiplex PCR tests for respiratory viruses is not yet fully determined. In one study of children with RSV infection, higher fever, longer hospital stays, and more frequent use of antibiotics were associated with multiple infections [
Two distinct advantages of the two-tube assay are the short assay time and the low cost. The assay requires 5-6 hours to complete 100 samples, including a half hour to prepare PCR mixture, the whole RT-PCR (3 hours), and the detection on the QIAxcel automatic electrophoresis (12 tests/15 minutes), while using other liquid chip-based assays might take an entire workday [
In conclusion, the two-tube assay developed in this study using automatic capillary electrophoresis is a rapid, cost-effective method with high sensitivity and specificity for the detection of respiratory virus infection, and it is demonstrated to have great potential for routine surveillance of respiratory virus infection.
The authors declare that they have no conflict of interests.
Jin Li was involved in the operation of the two-tube assay and designing of primers with Qin Meng, Xiumei Hu, and Hongwei Shen; Dr. Shunxiang Qi led the evaluation of the two-tube assay using the Seegene kit. Dr. Chen Zhang was responsible for providing the clinical samples and leading the evaluation of two-tube assay using the RVP method with Mrs. Mengjie Yang, Ji Wang, and Mrs. Miao Wang. Dr. Wenbo Xu offered great help in the supplement of different clinical samples as well as shared previous experimental data. Dr. Xuejun Ma designed and coordinated the study, analyzed data, and drafted the paper. Jin Li and Shunxiang Qi contributed equally to the study.
The authors are grateful to Dr. Brian Ring from Huazhong University of Science and Technology for critical review of this paper. This work was supported by the China Mega Project for Infectious Disease (2011ZX10004-001, 2012ZX10004-215, and 2013ZX10004-202).