Emerging Molecular Tests for Feline Toxoplasmosis: LAMP and RPA–CRISPR/Cas9

Rapid molecular diagnosis can be particularly valuable when conventional laboratory infrastructure is limited or when faster detection is required. For feline toxoplasmosis, newer approaches such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) combined with CRISPR/Cas9 are being explored as alternatives to conventional PCR. Their main appeal lies in simplified amplification conditions, rapid detection, and the potential for field-oriented use. 

LAMP: Molecular Detection Without Thermal Cycling 

LAMP amplifies DNA under constant-temperature conditions, typically at 60–65°C. The reaction uses four to six specially designed primers together with a strand-displacing DNA polymerase, such as Bst DNA polymerase¹. 

Unlike conventional PCR, LAMP does not require repeated cycles of denaturation, annealing, and extension. Large quantities of DNA can be generated within a short period, and amplification can be identified using fluorescent dyes, turbidity measurements, or visible white precipitates of magnesium pyrophosphate². 

This simpler amplification process gives LAMP considerable potential for veterinary diagnostic settings where access to conventional PCR equipment may be restricted. 

Targets and Detection Formats 

Several T. gondii-specific targets have been incorporated into LAMP approaches. One method targets the highly repetitive 529-bp sequence of T. gondii. Fluorescein isothiocyanate-labelled primers allow the amplification products to be detected using lateral flow dipsticks, producing a LAMP-LFD format with high specificity and sensitivity³. 

LAMP has also been adapted for detection of T. gondii DNA in cat feces. A colorimetric LAMP-RE approach targeting the B1 gene demonstrates how the technique can be adapted for parasite detection using a sample that can be collected without invasive tissue procedures¹. 

Another approach has incorporated recombinant proteins such as SAG1 and SAG2 into LAMP-based detection, with high sensitivity and specificity reported for T. gondii infection detection⁴. 

Important Limitation of LAMP 

Despite its advantages, LAMP results must be interpreted in the context of the infection stage. Detection in some LAMP approaches depends on circulating parasite DNA, meaning that the method may be limited in detecting infections beyond the early stage³. 

LAMP also should not be viewed as completely equipment-free. Although it avoids thermal cycling, the procedure still requires appropriate temperature control and technical handling. Nevertheless, its operational simplicity and rapid turnaround make it a promising option for settings where conventional molecular diagnostic infrastructure is restricted. 

RPA–CRISPR/Cas9: A Newer Diagnostic Strategy 

CRISPR/Cas9 is best known for its ability to recognize and cleave specific DNA sequences. The system uses the Cas9 endonuclease together with guide RNA (gRNA), which directs Cas9 toward the selected DNA sequence¹. 

Its diagnostic potential has been combined with recombinase polymerase amplification (RPA) to detect T. gondii DNA. One approach targets the highly repetitive 529-bp repeat element and uses lateral flow strips to visualize the detection result¹. 

A major advantage of RPA is that, unlike conventional PCR, it does not require thermal cycling^1,5. When paired with the sequence-specific DNA-cleavage capability of CRISPR/Cas9, the resulting platform provides a rapid and sensitive molecular detection approach. 

Potential for Veterinary Field Diagnostics¹ 

The combination of RPA and CRISPR/Cas9 is particularly interesting for veterinary medicine because the approach has the potential to simplify molecular detection and support field-deployable testing. Its reported characteristics make it relevant to low-resource environments and point-of-care diagnostic applications. 

However, CRISPR/Cas9-based detection remains at a developmental stage and is currently more closely associated with experimental and laboratory applications than established routine field diagnostics. 

Key Takeaways 

• LAMP amplifies T. gondii DNA at a constant temperature, avoiding conventional PCR thermal cycling. 

• B1 and the highly repetitive 529-bp sequence are important targets used in LAMP-based detection. 

• LAMP-LFD and colorimetric approaches provide simpler ways to visualize amplification results. 

• Some LAMP approaches may be limited by the availability of circulating parasite DNA during different infection stages. 

• RPA eliminates the need for thermal cycling and can be combined with CRISPR/Cas9 for sequence-specific detection. 

• RPA–CRISPR/Cas9 has potential for rapid, sensitive and field-oriented diagnosis, but its veterinary application remains under development. 

References 

1. Zhao D, Liao Y, Liu H, Wang J, Liang R, Zhou R, Ding J, Zhang S, Tang X. Comprehensive diagnostic approaches to feline toxoplasmosis: Bridging traditional methods and emerging technologies. Virulence. 2025 Dec 31;16(1):2563766. https://www.tandfonline.com/doi/pdf/10.1080/21505594.2025.2563766 

2. Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature protocols. 2008 May;3(5):877-82. https://www.academia.edu/download/73287951/nprot.2008.5720211021-9099-zgs498.pdf 

3. Xue Y, Kong Q, Ding H, Xie C, Zheng B, Zhuo X, Ding J, Tong Q, Lou D, Lu S, Lv H. A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs. Parasite. 2021 May 3;28:41. https://pmc.ncbi.nlm.nih.gov/articles/PMC8095094/pdf/parasite-28-41.pdf 

4. Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y. Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated isothermal amplification method using blood samples from patients. Journal of clinical microbiology. 2010 Oct;48(10):3698-702. https://journals.asm.org/doi/pdf/10.1128/jcm.00462-10 

5. Khan AH, Noordin R. Serological and molecular rapid diagnostic tests for Toxoplasma infection in humans and animals. European Journal of Clinical Microbiology & Infectious Diseases. 2020 Jan;39(1):19-30. https://pmc.ncbi.nlm.nih.gov/articles/PMC7087738/pdf/10096_2019_Article_3680.pdf