A field‐deployable insulated isothermal RT‐PCR assay for identification of influenza A (H7N9) shows good performance in the laboratory

Abstract Background Avian influenza A (H7N9) remains circulating in China. For countries at risk of introduction of H7N9, such as Vietnam, early detection of H7N9 virus is essential for the early containment of the virus. Insulated isothermal reverse transcriptase PCR (iiRT‐PCR) is a portable PCR system that can be deployed under field conditions to identify pathogens at the sampling site. Applying PCR at the sampling site will greatly reduce the time to obtain a diagnostic result which allows the veterinary authority to take immediate action to contain disease spreading. Objective To determine analytical and diagnostic sensitivity and specificity of the portable iiRT‐PCR for H7N9 virus detection. Methods A panel of 59 virus isolates, including H7N9, avian influenza viruses of subtype H1 to H13, swine and human influenza viruses, Newcastle disease virus, and infectious bursal disease virus, were tested by H7 and N9 iiRT‐PCR reagents, using probes and primers specific to H7 or N9, in comparison with laboratory‐based real‐time RT‐PCR assays to determine analytical sensitivity and specificity. Fifty oropharyngeal samples from experimentally infected chicken and ducks with H7N9 and 50 non‐infected control swabs were tested by the H7 iiRT‐PCR to determine diagnostic sensitivity and specificity. Results The H7 and N9 iiRT‐PCR reagents yielded comparable levels of analytical sensitivity and specificity with real‐time RT‐PCR for the detection of H7N9 virus. Diagnostic sensitivity and specificity of H7 iiRT‐PCR were 98% and 100%, respectively. Conclusion The observed high sensitivity and specificity of iiRT‐PCR for H7N9 detection show its potential for early detection of H7N9 in risk‐based surveillance.


| INTRODUC TI ON
The current avian influenza surveillance in Vietnam relies on the testing of samples at regional animal health offices (RAHO's) or the National Centre for Veterinary Diagnosis (NCVD). The average time to obtain a laboratory result is 2.5 days, with the majority of this time being attributed to sample shipment and transport to the laboratories (6-24 hours on average). Novel technologies, such as the insulated isothermal PCR (iiRT-PCR), are portable PCR systems that can be applied under field conditions by the fast PCR reaction (in 42 minutes). The iiRT-PCR portable system is a miniature portable device for field test, using freeze-dried thermostable reagents and powered by rechargeable lithium batteries. The iiRT-PCR applies the concept of Rayleigh-Benard convection to drive a PCR reaction in capillary tubes.
The primers/probe design rules are the same as for RT-PCR. While conventional PCR requires multiple cycles of heating and cooling, iiRT-PCR is performed through the creation of a temperature gradient in a capillary tube with a single heating source at the bottom of a capillary tube and establishment of thermal convection within the tube, mimicking the cycles of conventional real-time PCR. This system has recently been developed to rapidly detect pathogenic viruses, including white spot syndrome virus, classical swine fever, foot and mouth disease, equine influenza, bluetongue virus, and MERS-CoV. [5][6][7][8][9][10] Taking advantage of the iiRT-PCR portable system, the aim of this study was to validate the performance of the iiRT-PCR portable system for H7N9 detection.

| Viruses
This study used a panel of 59 virus isolates for analytical specificity that included 28 H7N9 AIVs, 7 H7 AIVs of Eurasian lineage but not in the cluster of recent H7N9 virus in China, 15 non-H7 AIVs, seven non-H7 influenza viruses of swine and human origin, two poultry viruses, Newcastle disease virus, and infectious bursal disease virus (Table 1).

Results:
The H7 and N9 iiRT-PCR reagents yielded comparable levels of analytical sensitivity and specificity with real-time RT-PCR for the detection of H7N9 virus.

Conclusion:
The observed high sensitivity and specificity of iiRT-PCR for H7N9 detection show its potential for early detection of H7N9 in risk-based surveillance.

K E Y W O R D S
diagnostic, influenza A (H7N9), PCR, sensitivity, specificity

| Clinical samples
A total of 50 oropharyngeal swab samples were collected from 25 chickens and 25 ducks experimentally infected with six different strains of AIV subtype H7N9. They were confirmed to be positive for AIV H7N9 by virus isolation and used as positive samples.
Another set of 50 oropharyngeal swabs were collected from the same chickens and ducks before infection. They were negative for AIV H7N9 by virus isolation and used as negative samples.

| In vitro transcribed RNA
In vitro transcribed RNA (IVT RNA) was generated from a plasmid con-

| POCKIT™ iiRT-PCR assay for AIV subtype H7 and N9
POCKIT™ iiRT-PCR assay for AIV subtype H7 was performed by using POCKIT™ influenza H7 reagent set (GeneReach USA). The N9 iiRT-PCR was developed by using the primers and probes in the RRT-PCR for avian influenza A (H7N9) described by WHO without any modifications. 11 The primers and probes used are listed in Table 2.
iiRT-PCR reagent is a single dose lyophilized format which can be shipped under ambient temperature for one week.
Briefly, the lyophilized reagent was reconstituted with 50 μL Premix Buffer B and mixed with 5 μL nucleic acid extract.
Subsequently, 50 μL of the final mixture was transferred to an R-tube

| Statistical analysis
Limit of detection 95% (LOD95%) of a reaction was determined by probit analysis at 95% confidence interval by SPSS v14 (SPSS). The 2 × 2 contingency tables were analyzed by kappa statistic using SPSS to determine the inter-assay agreement.
The analytical sensitivity of AIV H7 iiRT-PCR was further analyzed using 10-fold serial dilutions (up to 10 −8 ) of representative viruses from four groups of AIV H7N9 viruses. The detection endpoint (all triplicates positive) was compared to that of RRT-PCR assays using primers and probe set of H7 CODA and H7 CNIC. The sensitivity of AIV H7 iiRT-PCR was shown to be comparable to that of H7 CODA RRT-PCR assay in detecting the 4 representative H7N9 viruses, while having at least one log higher sensitivity when compared to that of H7 CNIC RRT-PCR assay ( Table 3). The sensitivity of AIV N9 iiRT-PCR was shown to be about one log lower than that of H7 CODA RRT-PCR assay in detecting the four representative H7N9 viruses, and sensitivity similar to that of H7 CNIC RRT-PCR assay (Table 3).

| Analytical specificity of iiRT-PCR for AIV subtype H7 and N9
The analytical specificities of the AIV H7 and N9 iiRT-PCR assay

| Diagnostic sensitivity and specificity of AIV H7 iiRT-PCR evaluated with oropharyngeal swabs from experimentally infected chickens and ducks
A total of 50 virus isolation-positive and 50 virus isolation-negative reference samples from chickens and ducks experimentally infected with different subtype H7N9 AIVs were tested by the H7 iiRT-PCR and compared in a 2 × 2 table (Table 4). Using virus isolation as the gold standard, the iiRT-PCR showed a sensitivity of 98% and a specificity of 100%. Results from field pilot studies (unpublished data) showed that it was feasible to install the system rapidly at any given sites and that personnel in the flied could be trained in the use of the iiRT-PCR system within 2 days. One of the challenges of the system is how it will be incorporated into current ongoing active surveillance designs. iiRT-PCR can be complementary to current surveillance designs, yet it needs to be clear if and how results will be confirmed by standard laboratory-based tests (real-time RT-PCR, virus isolation, or sequencing) and how data sharing will occur from local levels to province or central levels.

| D ISCUSS I ON
In conclusion, this study showed that field-based portable PCR (iiRT-PCR) can be used for the early diagnosis of H7N9 as an alternative approach to laboratory-based real-time PCR. Using field-based test will reduce the time to obtain a result and will enable possible quick response measures in the field, reducing the risk of further spread and human infections with H7N9 in currently non-infected countries.