Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

The overall goal of this procedure is to diagnose the occurrence of I myea species parasites in a chicken or chicken flock. This is accomplished by first preparing the reagents for loop mediated isothermal amplification or lamp reaction. Next, total genomic DNA is prepared from one or more intestinal sites postmortem.

Then the genomic DNA is added to the lamp master mix and incubated. Finally, the outcome of the lamp test is assessed. Ultimately, LAMP is used to determine the occurrence of specific I miria species to improve stock management and as a tool for diagnosis of disease.

So the main advantage of this technique over traditional methods like morphological microscopy or PCR as it is less subjective and it doesn't require expensive laboratory facilities. Using this approach can improve chicken health and welfare by improving diagnosis of Arian infection. To prepare the template for the Erea lamp ase, choose the section of intestine to be tested.

Use this table as a guide to sample site selection and the eria species most likely to be present. This is of key importance since each species that infects chickens is defined by the intestinal region that it targets using sterile scissors or a scalpel. Excise five centimeter or longer lengths of the intestinal sections selected to test for eria.

Cut the sample open longitudinally. Remove most intestinal contents and use the edge of a sterile glass microscope. Slide or sterilize scissor blade to scrape free the cells from the mucosal layer.

Put the scraped material into a sterile 1.5 milliliter screw top micro centrifuge tube containing 100 microliters of sterile TE with 10%weight, but volume cell X 100 resin and firmly screw on the top. Shake the samples vigorously for one minute, then incubate in a boiling water bath for 10 minutes. After boiling, allow each sample to cool at the ambient temperature for one to two minutes.

Centrifuge the samples at 10, 000 times G for one minute before collecting two microliters of the supernatant per lamp assay to be used as a template to prepare I miria lamp primer stocks adequate for 100 assays. Reconstitute each lyophilized I miria lamp primer by adding molecular grade water to a concentration of 100 micromolar pipette 60 microliters of molecular grade water into a separate 0.5 milliliter flip top micro centrifuge tube for each iria species to be assay. Using the volume shown in this table.

Add primers, F-I-P-B-I-P-F three B three LF and LB specific to the target eria species to the water. Creating a series of seven species specific primer mixes. Briefly invert the primer solutions to mix them, them pulse micro fuge, and freeze until required.

Using the volumes listed in this table and the guidelines in the text protocol, prepare a lamp reaction master mix for each iria species to be assay to carry out the lamp assay pipette 23 microliters of IEA species specific B-S-G-D-N-A polymerase lamp master mix into a 0.5 milliliter micro centrifuge tube. Add two microliters of previously prepared DNA template making a final reaction volume of 25 microliters as a positive control. Add two microliters of eria species specific genomic DNA to one reaction.

Add two microliters of molecular grade water to a reaction as a negative control incubating a water bath or heat block at 62 degrees Celsius for 30 minutes. Optionally deactivate the B-S-T-D-N-A polymerase by heating to 80 degrees Celsius for 10 minutes. If the reaction is not going to be read immediately at the conclusion of the incubation under indoor light, assess the color of each reaction by eye.

Negative results will appear pink to violet in color and positive results will appear. Sky blue optionally. To confirm the lamp assay result, mix five microliters of lamp reaction product with one microliter of DNA gel loading buffer and run on a prestain 2%ARO gel.

Use a one killer base molecular sized DNA ladder to determine fragment size testing three. Birds collected from a US broiler farm as part of a surveillance program Yielded three sets of intestinal samples as shown here. Targeted application of the species specific lamp assays and prioritizing the preferred intestinal sites for each I miria.

Species allowed visual identification of an irian infection in all birds tested using hydroxy natal blue as an indicator. The color achieved with a negative lamp reaction when using hydroxy can range from violet to pink, but is always distinct from the blue achieved by a positive result. Confirmation by aose gel electrophoresis provided comparable results as shown here.

Preparing the DNA from one pulled sample per bird representing material collected from each of the specific intestinal sites and tested with all seven lamp assays provided the same result as when each intestinal site was processed separately Once mastered, this technique can be performed in 45 minutes if it is done properly following this procedure. Other techniques like lesion scoring can also be performed in order to assess overall intestinal health.