Multiplex Real-Time PCR Assay for Intestinal Parasite Detection
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Solution Overview
Problem
Current methods for detecting intestinal parasites, such as microscopy and immunodiagnosis, are inefficient, prone to misjudgments, and lack specificity, especially in differentiating between closely related species, and existing PCR assays face challenges with PCR inhibitors and amplification biases in stool samples.
Innovation Solution
A multiplex real-time PCR assay using specifically designed primers and probes targeting conserved sequences in intestinal parasites, allowing for simultaneous amplification and detection of multiple species in a single reaction, while optimizing amplicon design to overcome PCR inhibitor effects and ensure equal amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If microscopy is used for parasite detection, then equipment simplicity is maintained, but diagnostic accuracy and species differentiation capability deteriorate
Solution Approach 1:
The patent replaces the mechanical/optical microscopy system with a molecular biology-based PCR system. Instead of visually examining stool samples under a microscope, the invention uses polymerase chain reaction to amplify and detect parasite-specific DNA sequences, providing superior diagnostic accuracy and species differentiation while accepting the increased complexity of molecular techniques
Solution Approach 2:
The patent introduces DNA amplification as an intermediary step between sample collection and detection. By amplifying parasite-specific DNA sequences before detection, the method enables highly accurate identification of parasite species that cannot be distinguished by microscopy alone, serving as a bridge between simple sampling and precise diagnosis
2Measurement precision
If multiple parasite species are detected using individual PCR reactions, then detection specificity is improved, but testing time and operational complexity increase
Solution Approach 1:
The patent combines multiple species-specific PCR reactions into a single multiplex PCR reaction. By including multiple primer pairs targeting different parasite species in one reaction vessel, the method maintains high species detection specificity while dramatically reducing the number of separate tests required, thereby improving productivity and reducing operational complexity
Solution Approach 2:
The patent creates a universal detection platform that can identify multiple parasite species simultaneously through a single assay. The multiplex PCR system is designed to detect various intestinal parasites using one reaction protocol, making the method broadly applicable and highly efficient for clinical diagnostics
3Reliability
If standard PCR assays are used on stool samples, then detection capability is achieved, but PCR inhibitor interference reduces amplification efficiency
Solution Approach 1:
The patent optimizes PCR reaction parameters specifically for stool samples containing inhibitors. By adjusting MgCl2 concentration, primer concentrations, and cycling conditions, the method achieves reliable amplification despite the presence of PCR inhibitors in fecal matter, maintaining both detection reliability and amplification efficiency
4Measurement precision
If conventional immunodiagnosis is used, then detection speed is improved, but sensitivity and specificity deteriorate
Solution Approach 1:
The patent replaces immunodiagnostic methods with molecular-based PCR detection. Instead of using antibodies to detect parasite antigens, the invention uses DNA amplification to detect parasite genetic material, achieving superior sensitivity and specificity while maintaining rapid turnaround time through efficient PCR protocols
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method provides rapid, specific, and sensitive detection of multiple intestinal parasite species, improving diagnostic accuracy and efficiency, reducing the need for antigen testing and microscopy, and enhancing infection control by enabling differentiation between species.
Implementation Method 1
contacting the sample or nucleic acid isolated therefrom with oligonucleotide primers in an amplification assay to provide a reaction mix for nucleic acid amplification
Implementation Method 2
performing a nucleic acid amplification reaction with the reaction mix obtained from step i) comprising DNA from the biological sample as a template, so that the target sequences of the intestinal parasite(s) is/are specifically amplified
Implementation Method 3
detecting the presence of an amplified target sequence in the reaction mix, wherein the presence of the target sequence is indicative of the presence of intestinal parasites in the sample
Data Source
AI summary
This invention relates to the field of detection of intestinal parasites from patient, food or environmental samples, preferably from a stool sample. Particularly, the present invention provides a polymerase chain reaction (PCR) based assay method for detection of intestinal parasite infection, particularly the infection of parasite species selected from a group consisting of Hymenolepis nana, Hymenolepis diminuta, Fasciolopsis buski, Encephalitozoon spp. (such as E. intestinalis, E. cuniculi and E. hellem), Enterocytozoon bieneusi, Enterobius vermicularis, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Schistosoma mansoni, Blastocystis hominis, Ancylostoma duodenale and liver worms, such as Clonorchis sinensis, Opisthorchis spp., and Metorchis spp. The present invention further provides materials such as primers, primer pairs and probes for use in the method of the invention. Preferably, the method of the invention is a multiplex real-time PCR assay for rapid determination of clinically important intestinal parasites.