2012-11-05-世界卫生组织-Mutant_Analysis_by_PCR_and_Restriction_Enzyme_Cleavage_MAPREC_for_Oral_Poliovirus_Sabin_Vaccine_Types_1,_2_or_3_34页_692kb

MAPREC SOP Summary: Mutant Analysis for Oral Poliovirus Vaccine

Purpose

The MAPREC assay (Mutant Analysis by PCR and Restriction Enzyme Cleavage) is a WHO-recommended standard operating procedure to quantify single base mutations in poliovirus vaccine strains, ensuring safety by detecting potential reversion to neurovirulence. It is critical for types 1, 2, and 3 Sabin vaccines.

Key Objectives

• Determine the proportion of revertant mutations in viral RNA.
• Flag batches where mutation levels exceed acceptable thresholds for non-acceptability in vaccines.
• Assess production consistency and vaccine quality.

Assay Overview

1. Molecular Procedure:

   • Extract RNA from vaccines or isolates.
   • Synthesize cDNA using reverse transcriptase.
   • Amplify viral RNA segments with specific PCR primers targeting mutation sites (e.g., base 480 for type 1, base 481 for type 2, base 472 for type 3).
   • Ligate a unique restriction enzyme site during labeling.
   • Digest products with specific enzymes, separate fragments via polyacrylamide gel electrophoresis, and quantify bands using radioactive or fluorescent detection.
   • Calculate revertant content percentage using formulas like ( \text{% Revertants} = \left( \frac{\text{DL}}{\text{DU} + \text{DL}} - \frac{\text{CL}}{\text{CU} + \text{CL}} \right) \times 100 ) for types 2 and 3, or similar for type 1 with additional digests.

2. Validations and Criteria:

   • Each test includes controls (cDNA water, PCR blanks, DNA standards) to detect contamination.
   • Within-assay variability must be low (e.g., standard deviation ≤ 0.3), and results consistent with previous tests using IS DNAs or comparison standards.
   • Minimum acceptable mutant thresholds are based on WHO recommendations (e.g., >0.9% for type 3 base 472 mutation).
   • Up to 5 individual determinations per batch, with repeats, and strict monitoring for consistency.

Critical Aspects

• Reagents: High-purity primers and restriction enzymes are essential; enzyme efficacy and specificity must be validated.
• Controls: Include HMVR, LMVR, IS DNA, and PCR cDNA controls to ensure assay reliability.
• Contamination Prevention: Dedicated areas, equipment, UV sterilization, and pipette protocols to avoid cross-contamination.
• Gels: Use 10% polyacrylamide gels for separation, with optimized labeling and quantification via specialized software for accurate band intensity measurement.

Significance

• Prevents vaccine reversion to virulence by monitoring mutations in the 5' non-coding region.
• Aligns with WHO quality guidelines for oral poliovirus vaccines, supporting global polio eradication efforts.
• Ensures consistency in vaccine production and batch acceptability decisions based on mutation levels.