2013-04-07-世界卫生组织-Real-time_RT-PCR_Protocol_for_the_Detection_of_Avian_Influenza_A_H7N9_Virus_6页_94kb

Real-time RT-PCR Protocol for the Detection of Avian Influenza A(H7N9) Virus Summary

Core Content

This document provides a real-time RT-PCR (rRT-PCR) protocol for the detection of avian influenza A(H7N9) virus, developed by the WHO Collaborating Centre for Reference and Research on Influenza at the Chinese National Influenza Center. The protocol is intended for emergency use and is not for commercial development or profit.

Main Purpose

The primary goal of the protocol is to specifically detect the avian influenza A(H7N9) virus by targeting the matrix (M), hemagglutinin (H7), and neuraminidase (N9) genes using real-time RT-PCR. It also includes a housekeeping gene (RnaseP) and a control gene (FluA) for internal control and typing purposes.

Materials and Equipment

• Real-time fluorescence quantitative PCR analysis system
• Bench top centrifuge for 1.5 mL Eppendorf tubes
• 10, 200, and 1000 μL pipettors with plugged tips
• Vortex mixer
• QIAGEN RNeasy Mini Kit
• AgPath one-step RT-PCR kit
• Specific primers and probes for H7, N9, M, and RnaseP genes
• RNase-free 1.5 mL Eppendorf tubes and 0.2 mL PCR tubes
• Powder-free disposable latex gloves, goggles, headgear, shoe covers
• β-thioglycol and 70% alcohol

Biosafety Precautions

• Specimen lysis should be conducted in a BSL-2 facility with BSL-3 level personal protection equipment.
• Subsequent procedures can be carried out in a BSL-2 laboratory with designated areas: reagent preparation, specimen preparation, and amplification/detection.
• The workflow must follow a clean-to-dirty direction to prevent cross-contamination.

Procedure Overview

4.1 Nucleic Acid Extraction

• Extract RNA in a BSL-2 biohazard hood using the manufacturer's instructions.
• Elute RNA in a final volume of 50 μL H₂O.

4.2 Quality Control Parameters

• Negative control: Sterile water extracted under the same conditions as the specimens.
• Reagent blank control: RNase-free H₂O.
• Positive control: RNA of the A(H7N9) virus.
• Internal positive control: Ribonucleoprotein (RNP) is recommended.

4.3 Reaction System Preparation

Each reaction mixture is prepared as follows:

4.4 Reaction Setup

• Aliquot 20 μL of the reaction mixture into 0.2 mL PCR tubes or a 96-well PCR plate.
• Label tubes clearly.

4.5 Loading and Cycling

• Add 5 μL of template RNA (for negative control, test specimens, or positive control) into the respective tubes in a BSL-2 biohazard hood.
• Use the following cycling program:

1. 45°C for 10 minutes
2. 95°C for 10 minutes
3. 95°C for 15 seconds
4. 60°C for 45 seconds

Repeat steps 3 and 4 for 40 cycles.

4.7 Result Analysis

• Negative result: Ct value is undetectable.
• Positive result: Ct value ≤ 38.0.
• Repeat test: Specimens with Ct > 38.0 should be repeated. If the repeat result is the same, it is considered positive; otherwise, negative.

4.8 Quality Control Criteria

• Negative control must be negative.
• Positive control Ct value should not exceed 28.0.
• If these criteria are not met, the test is considered invalid.

Troubleshooting

• False positives: May result from environmental contamination. Ensure strict unidirectional workflow, ventilate the lab, clean the workbench, autoclave tubes and tips, and use fresh reagents.
• RNA degradation: If the positive control Ct value exceeds 30, check for RNA degradation and ensure all materials are RNase-free.

Cautions

• Add negative control first, then the specimen, and finally the positive control to avoid cross-contamination.
• Use dedicated equipment and materials for each area (e.g., lab coats, pipettors, tips, gloves).
• Regularly maintain and calibrate the incubator, PCR cycler, and pipettors every 6 months.

Protocol Use Limitations

• This protocol is optimized for the AgPath one-step RT-PCR kit and may not be compatible with all systems.
• It is designed for use on 96-well format thermocycler systems such as Stratagene QPCR instruments (MX3000® or MX3005®).