Primer & Probe Design for RPA

RPA Primer & Probe Design

Primer & Probe Design for RPA

Complete target sequence input, parameter configuration, and candidate site filtering in the browser. No local client installation or environment setup is required, so you can focus on experimental design.

Input Sequence

Desktop is recommended. Mobile has lower result readability and no History feature.

Product Introduction

RPA Primer & Probe Design is a web-based tool for automated design of RPA primers and probes. Built on the PrimedRPA algorithm, it supports both single-sequence and multi-sequence analysis to help researchers rapidly identify candidates suitable for experimental validation.

Designed for pathogen detection, molecular diagnostics, food safety, and environmental monitoring applications, the platform combines conservation analysis, primer quality assessment, and background specificity screening to improve design efficiency and assay reliability.

  • Designed for High-Diversity Pathogen Scenarios

    Supports multi-sequence input (up to 10 sequences; extra entries are truncated) and extracts conserved target regions.

  • Automated Primer & Probe Design

    Automatically generates candidate RPA primers and probes from target sequences.

  • EXO & NFO Compatibility

    Supports common probe formats for fluorescence and lateral-flow RPA assays.

  • Conserved Region Analysis

    Supports multi-sequence input and automatically identifies conserved target regions for primer and probe design.

How to use?

STEP 1

Input target sequences (paste FASTA text or upload files).

STEP 2

Configure General / Cpf1 Setting / Primers Options parameters based on experiment needs.

STEP 3

Click "Start Design" to submit the task and begin automatic computation.

STEP 4

After completion, view results in a new tab and download full files.

Parameter Details

Input

  • Input Sequence

    Input target sequences to design against (FASTA supported). If both text and file are provided, text input takes priority.

  • Upload FASTA File

    Use when target sequences are long or from external files. Standard .fa/.fasta/.txt files are recommended.

Options

  • Probe Required

    Controls whether probes are co-designed and probe type selection (NO/EXO/NFO), affecting final recommendation paths and result structure.

  • Desired Primer Length

    Defines primer length range or fixed length. Too short may reduce specificity; too long may reduce amplification efficiency.

  • Desired Probe Length

    Defines probe length range or fixed length to balance binding stability and detection sensitivity.

  • Max Amplicon Length

    Sets maximum amplicon length. In general, shorter products provide faster amplification and better detection stability.

  • Repeat Nucleotide Cut-off

    Limits continuous repeats of the same nucleotide to reduce nonspecific risks caused by low-complexity regions.

  • GC Range (Min / Max)

    Controls GC content range for candidate sequences. Too low may weaken binding stability; too high may increase secondary structure risk.

  • Maximum Number of Sets

    Controls the maximum number of output candidate sets to balance coverage and downstream screening cost.

Advanced Setting

  • Input FASTA Classification

    Declares input sequence type (single sequence / multiple unaligned / multiple aligned), affecting internal parsing and alignment strategy.

  • Primer/Probe Identity Threshold (%)

    Homology threshold. Higher values enforce stricter conservation requirements, mainly used for consistency control in multi-sequence inputs.

  • Dimerisation Threshold (%)

    Controls dimerization risk threshold to filter candidates with overly strong potential complementary binding.

  • Background Cross-reactivity Threshold (%)

    Background cross-reactivity threshold for filtering candidates with excessive similarity to background sequences. Editable only when background input (text or file) is provided.

  • Background Hard Fail Filter

    Whether to enable strict background filtering. When enabled, high-risk candidates are removed more aggressively. Editable only when background input (text or file) is provided.

  • Blastn Cross Reactivity Search Settings

    Background search intensity (Basic/Advanced/Fast), used to balance compute speed and filtering granularity. Editable only when background input (text or file) is provided.

  • Blastn Evalue

    Background alignment significance threshold. Smaller values are stricter and usually reduce background-hit tolerance. Editable only when background input (text or file) is provided.

Background Check

  • Background Sequence

    Optional background DNA sequences for off-target risk exclusion. If omitted, strict background filtering is skipped.

  • Upload Background FASTA

    Uploading files is recommended when many background sequences are used, enabling complete cross-reactivity evaluation.

Parameter Tuning Suggestions

When No Results Are Returned (Relax First)

  • IdentityThreshold (e.g., 99 -> 95)
  • DimerisationThresh (moderately increase)
  • CrossReactivityThresh (moderately increase)
  • AmpliconSizeLimit (moderately increase)

When Results Are Too Many or Computation Is Slow (Tighten First)

  • Narrow PrimerLength / ProbeLength ranges
  • Reduce MaxSets
  • Use Fast for background sensitivity

Calculation Principle

STEP 1

Input and Preprocessing

01The service receives target sequences (InputSequence or InputFile).
02Parameters include primer/probe length, GC range, homology threshold, repeated nucleotide limit, amplicon size limit, dimer threshold, and background filtering thresholds.
03ProbeRequired determines probe workflow: NO performs FP/RP only; EXO/NFO performs FP/RP + Probe.

STEP 2

Alignment Summary Construction

01If input is multi-sequence (MS), sequences are first unified on a shared alignment coordinate.
02For each position, consensus nucleotide (most common nucleotide) and identity score (IdentityScore) are calculated.
03A full-length alignment summary is generated, and downstream candidates are produced by sliding windows over this positional score map.

STEP 3

Candidate Oligonucleotide Scanning (Primer/Probe Candidate Generation)

01Start positions are scanned by candidate type (Primer/Probe) and length (single value or range), generating candidate sequences window by window.
02Each candidate records start position, length, mean identity, 5'/3' conserved anchor lengths, GC%, and self-dimer/structure score.

STEP 4

Candidate Filtering (Single-Sequence Level)

01Homology filtering: IdentityScore >= IdentityThreshold.
02GC filtering: MinGC < GC < MaxGC; repeated nucleotide filtering: no repeats longer than NucleotideRepeatLimit.
03Self-complementarity/secondary structure filtering: score must not exceed DimerisationThresh.
04Probe-specific filtering: EXO enforces specific motif/site rules.
05Background cross-reactivity filtering (optional): BLAST against background libraries plus advanced cross-reactivity scoring; candidates above CrossReactivityThresh are removed. When HardCrossReactFilter != NO, hard-fail primers are removed directly.

STEP 5

Combination Search (FP/RP/Probe)

01Combinations (FP/RP/Probe) are assembled from candidates that passed single-candidate filtering.
02Combination constraints: amplicon length must not exceed AmpliconSizeLimit; probe insertion rules apply when probe is enabled; combinations with pairwise interaction scores above thresholds are removed.
03Passing combinations record FP/RP/Probe sequences and positions, amplicon size, max dimer score, and max background cross-reactivity score (if background check is enabled).

STEP 6

Result Ranking and Trimming

01Results are ranked ascending by Max Dimerisation Percentage Score (lower is better).
02Final outputs include result tables and downloadable packages (alignment summary, candidate sites, and final combinations).

Open-Source Project Reference

Project：PrimedRPA: RPA Primer and Probe Set Finder - Higgins M et al. Submitted. 2018 - GPL-3.0

Project URL：https://github.com/MatthewHiggins2017/bioconda-PrimedRPA

Compliance Note (Current Deployment)：Current deployment executes PrimedRPA server-side and returns results via API, without distributing PrimedRPA program copies to end users. Under this usage pattern, GPL source-distribution obligations are not triggered.

Frequently Asked Questions

Q1: Result shows: Input sequence can not be empty.

Cause: neither InputSequence nor InputFile was provided. Fix: one of them is required.

Q2: What input scale is supported?

Multi-sequence input is supported (set Input fasta classification to MS or MAS), up to 10 sequences total; each sequence supports up to 1000 bases. Extra sequences are discarded automatically. Exceeding per-sequence base limits will trigger errors.

Q3: Result shows: Input file must be UTF-8 text.

Cause: uploaded file encoding is not UTF-8. Fix: convert to UTF-8 and upload again.

Q4: Result shows: Sequence contains invalid characters. Only A/C/T/G/U are allowed.

Cause: sequence includes non-ACTGU characters (e.g., numbers, symbols, extended letters). Fix: clean invalid characters and retry.

Q5: Result shows: Sequence length must be less than 1000.

Cause: sequence length exceeds backend limit. Each sequence segment supports up to 1000 bases.

Q6: Result shows: Alignment file error length of all sequences is not equal

Cause: in multi-sequence mode (MS), sequence lengths are inconsistent. Fix: run standard MSA first and ensure equal lengths before submission.

Q7: Result shows: No Oligos Passed Filtering

Cause: all candidates were filtered out at single-sequence filtering stage. Common triggers: overly strict IdentityThreshold, too narrow GC range, too strict NucleotideRepeatLimit, too strict DimerisationThresh, or strict background thresholds. Fix: gradually relax constraints (priority: Identity, GC, Dimer, CrossReactivity).

Q8: Result shows: No valid primer-probe combinations found

Cause: single candidates exist, but all combinations fail. Common triggers: too small AmpliconSizeLimit, mandatory probe mode reducing combination space, or interaction scores exceeding thresholds. Fix: moderately increase AmpliconSizeLimit or relax dimer/length ranges.

Q9: Result shows: PrimedRPA timed out after N seconds

Cause: task did not finish before timeout (more likely with long sequences, wide parameters, or large background libraries). Fix: reduce search space (MaxSets, length ranges, background sensitivity) or increase timeout limits.

Q10: Result shows: RPA processing failed

Cause: uncategorized runtime exception (dependency tool errors, insufficient resources, temp-file issues, etc.). Fix: provide task_id / request_id to backend for log troubleshooting.

Q11: Result shows: QUEUED — what does it mean?

Server resources are limited. When too many tasks run simultaneously, new tasks wait until earlier tasks complete before execution starts.

Q12: What is the difference between single-sequence and multi-sequence modes?

Single-sequence mode (SS) designs directly on input sequence; multi-sequence mode (MS) aligns sequences on a unified coordinate first, then generates candidates based on conserved sites. For multi-sequence input, ensure sequence quality and formatting consistency.

Q13: Why is background sequence input recommended (Background Check)?

Background filtering can significantly reduce cross-reactivity risk against related non-target sequences. In high-homology scenarios (e.g., same-genus pathogens), enabling background checks and setting CrossReactivity thresholds properly is strongly recommended.

Q14: Why are some background-related parameters disabled?

When no background content is provided (text or file), background filtering parameters are automatically disabled and system default placeholder values are used. They become editable only after background content is provided.

1-on-1 Expert Support

Get personalized guidance from our technical experts to help solve complex research challenges, so you can stay focused on your science.

01

Chat with us

Send us an email: info@ezassay.com

02

Call us

Call our team Mon–Fri from 8am to 5pm.

+86 19925271988

Submit your information and we will contact you as soon as possible.