Testing Methods & Technology¶

SENO employs a comprehensive suite of molecular biology techniques optimized for avian sample types. Our self-developed reagents are designed for the specific challenges of feather and swab samples, including melanin inhibition resistance and low-template (low-copy number) amplification. All methods are validated following CLSI EP guidelines and operate under our ISO 9001:2015 and ISO 13485:2016 certified quality management systems.

Core Technologies¶

Method	Application	Key Advantage	Link
Conventional PCR	DNA sexing, genotyping	Cost-effective, gel-visible results	Details →
qPCR / Fluorescent PCR	Pathogen detection, quantification	Real-time Ct values, higher sensitivity	Details →
Capillary Electrophoresis	Fragment analysis, fingerprinting	Base-pair resolution of alleles	Details →
Reagent Development	Custom primer/probe design	Species-optimized assay performance	Details →

Method Selection Guide¶

The choice of testing method depends on the specific diagnostic or analytical question. The table below provides a quick-reference guide for common testing scenarios.

Testing Scenario	Recommended Method	Sample Type	Turnaround
Determine bird sex	Conventional PCR (P2/P8) + gel	Plucked feathers (3–5)	1–2 days
Screen for PBFD	qPCR (TaqMan)	Swab or feather	1–2 days
Screen for APV / Polyomavirus	qPCR (TaqMan)	Swab or feather	1–2 days
Investigate disease outbreak	Multiplex qPCR panel	Oral + cloacal swab	2–3 days
Racing pigeon performance	SNP analysis (PCR + CE)	Feather or blood	3–5 days
Theft recovery / ID	Microsatellite CE (12 loci)	Feather or blood	3–5 days
Parentage confirmation	Microsatellite CE + analysis	Feather or blood from parent + offspring	3–5 days
Virology research	qPCR or conventional PCR + sequencing	Feather, swab, or tissue	5–7 days

Technology Comparison¶

Each technology has distinct performance characteristics that make it suitable for particular applications. The following comparison highlights the key differences across our four core methods.

Feature	PCR	qPCR	CE	Reagent R&D
Quantification	No	Yes (Ct)	Relative (peak height)	N/A
Sensitivity	~100 copies	~10 copies	~50 copies	Design-dependent
Resolution	~10 bp (gel)	1 Ct (~2×)	1 bp	N/A
Time to result	3–4 h	1.5–2 h	4–5 h	2–4 weeks
Cost per reaction	Low	Medium	Medium-high	Variable
Multiplex capacity	2–4 targets	4–6 targets	10–20 markers	Unlimited
Post-PCR handling	Required (gel)	Not required	Required (plate prep)	N/A
Automation level	Semi-automated	Fully automated	Fully automated	Manual

Validation Framework¶

Every testing method at SENO undergoes a standardized validation process before being deployed for clinical or diagnostic use. Our validation framework follows CLSI EP guidelines — the same standards used by human molecular diagnostics laboratories.

CLSI Guideline	Application	Key Metrics
EP05 — Precision	Intra- and inter-assay reproducibility	SD, CV (< 3% for Ct, < 5% for fragment size)
EP06 — Linearity	Dynamic range of qPCR assays	R² ≥ 0.98, efficiency 90–110%
EP07 — Interference	Melanin, hemoglobin, heparin resistance testing	Ct shift < 1.5 cycles
EP12 — Qualitative Performance	Clinical sensitivity / specificity	≥ 95% agreement with sequencing
EP15 — User Verification	Method verification in SENO's lab	Pass/fail against manufacturer claims
EP17 — Detection Limits	Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantification (LoQ)	LoD ≤ 10 copies/reaction (qPCR)

For detailed validation data, see Accuracy Validation and PBFD qPCR Validation.

Sample Compatibility¶

Our methods are validated for a broad range of sample types commonly submitted for avian testing. Each sample type presents unique challenges that our protocols and reagents are designed to address.

Sample Type	Compatible Methods	Key Challenge	SENO Solution
Plucked feather follicle	PCR, qPCR, CE	Low DNA yield, melanin inhibition	Optimized lysis buffer + melanin-resistant PCR mix
Oral / cloacal swab	PCR, qPCR	Inhibitors from mucus, bacteria	Stabilization buffer with inhibitor removal
Blood (EDTA)	PCR, qPCR, CE	Hemoglobin interference	Purification step + amplification enhancers
Blood (FTA card)	PCR	Low input, storage degradation	Small-punch protocol with extended extension time
Tissue (post-mortem)	PCR, qPCR, CE	DNA degradation, PCR inhibitors	Short-amplicon design (< 200 bp)
Eggshell membrane	PCR	Very low DNA yield	Nested PCR approach
Feather (non-follicle)	PCR (limited)	No genomic DNA, keratin	Not recommended — use follicle-containing feathers

Quality Control Integration¶

All testing methods are governed by SENO's multi-tier quality control framework, which is documented in detail in the Quality Control & SOPs section. Key QC elements that apply across all methods include:

QC Element	Frequency	Method Applicability
Positive controls	Every batch	PCR, qPCR, CE
Negative controls	Every batch	PCR, qPCR, CE
No-template control (NTC)	Every batch	PCR, qPCR, CE
Extraction blank	Every batch	PCR, qPCR
Internal amplification control	Every qPCR reaction	qPCR only
Ladder / size standard	Every CE injection	CE only
Inter-operator comparison	Quarterly	All methods
External proficiency testing	Annually	All methods

Method Development Pipeline¶

New testing methods are developed through SENO's structured R&D pipeline, from concept through to clinical deployment.

Phase	Activities	Duration	Deliverable
1 — Feasibility	Literature review, in-silico primer design, BLAST analysis	1–2 weeks	Design report
2 — Optimization	Gradient PCR, annealing temp matrix, MgCl₂ titration	2–4 weeks	Optimized protocol
3 — Validation	CLSI EP studies (precision, sensitivity, specificity)	4–8 weeks	Validation report
4 — Verification	50+ clinical samples, comparison with reference method	2–4 weeks	Verification report
5 — Deployment	SOP drafting, technician training, QC target setting	1–2 weeks	Released SOP

Cross-References¶

PCR Technology → — Detailed conventional PCR methodology
qPCR / Fluorescent PCR → — Real-time PCR detection chemistry
Capillary Electrophoresis → — Fragment analysis and DNA fingerprinting
Reagent Development → — Self-developed reagent design and manufacturing
Laboratory Workflow — End-to-end sample processing pipeline
Quality Control & SOPs — QC framework governing all testing methods

Frequently Asked Questions¶

Q: Which method is best for routine sexing? A: Conventional PCR with P2/P8 primers followed by agarose gel electrophoresis. It is cost-effective, reliable (> 99% concordance with known-sex birds across 50+ species), and requires no specialized instrumentation beyond a thermocycler and gel system.

Q: When should qPCR be used instead of conventional PCR? A: qPCR is preferred when quantification is needed (viral load for disease monitoring), when higher sensitivity is required (detecting early or low-level infections), or when minimizing contamination risk from post-PCR handling is critical (closed-tube detection).

Q: What is capillary electrophoresis used for in avian testing? A: Capillary electrophoresis enables base-pair resolution fragment sizing, which is essential for DNA sexing fragment analysis (distinguishing CHD-W and CHD-Z amplicons that may differ by only 10–30 bp) and microsatellite-based DNA fingerprinting for individual identification and parentage verification.

Q: Does SENO develop its own reagents? A: Yes. SENO designs and manufactures proprietary PCR master mixes, qPCR probe sets, DNA extraction kits, primer panels, and stabilization buffers. These reagents are specifically optimized for avian sample types and are produced in our ISO 13485-certified facility.

Q: Are all methods validated? A: Yes. Every testing method undergoes full CLSI EP-compliant validation before deployment. Validation data — including sensitivity, specificity, precision, and accuracy — is documented and maintained per method. See Accuracy Validation for details.

This page provides an overview of SENO's testing methods portfolio. For detailed protocols and SOPs, refer to the individual method pages and the Quality Control section. For inquiries about method validation data, contact [email protected].