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Molecular methods are used to identify insect vectors, (Hadi M.H. Al-Mayali*, 2017), pathogens, (Azizi, Askari, Kalantari, & Moemenbellah-Fard, 2016), and their hosts (source(s) of the blood meals), (Abbasi, Cunio, & Warburg, 2009). Most of what I found specific to sand flies were Polymerase Chain Reaction (PCR) based methods. PCR is a laboratory technique that allows researchers to amplify specific bits of target DNA. It can be the DNA of the vector, the pathogen, or of the host species, (Kent, 2009). PCR relies on a thermo cycler to control the temperature of the reaction, triggering each stage of the PCR, denaturing, annealing, and extension of the target DNA segments, (Garibyan & Avashia, 2013). There are several different ways to do PCR, such as restriction fragment length polymorphism (RFLP PCR), which uses a restriction enzyme to find Single Nucleotide Polymorphisms (SNP’s) for a particular pathogen, vector, or host. The DNA region is amplified using PCR then the amplicon is cut. The results are “read” on a gel after gel electrophoresis. This method has been useful in the identification of the host (meal) of the sand fly, (Kent, 2009). RFLP PCR is useful because it is accurate and fast, (Tiwary, Kumar, Rai, & Sundar, 2012). The DNA region is amplified using PCR then the amplicon is cut. The results are “read” on a gel. Similarly, Amplified Fragment Length Polymorphism (AFLP PCR) can be used to evaluate DNA sliced at specific restriction sites. These sites may be varied between species. Real Time PCR (rtPCR) is amplification of specific target DNA sequences that are tagged with a florescent flag so the amplification can be seen in real time, (Nayduch, Fryxell, & Olafson, 2019). Heteroduplex analysis is a PCR method in which the DNA is denatured then re-annealed much more slowly than normal, (Kent, 2009). This causes each target gene sequence to amplify 4 times (creates 4 copies of the double stranded DNA), (Kent, 2009). Double stranded DNA from an individual contain homoduplexes and heteroduplexes of DNA from the locus in which the two alleles reside, (Kent, 2009). This method is also used in analyzing vector hosts, (Kent, 2009). PCR was used to identify sand flies in Iraq using the traditional method, (Hadi M.H. Al-Mayali*, 2017). PCR combined with reverse line-blot hybridization is also a accurate and fast way to identify hosts of sand fly vectors, (Abbasi et al., 2009). It is done on a reusable nylon membrane and a researcher can run more than one membrane at a time (high through-put), (O’Sullivan, Zhou, Sintchenko, Kong, & Gilbert, 2011). It is possible to find up to 43 gene targets in as many samples using only one reaction, (O’Sullivan et al., 2011). This method is also cost effective, costing only $2 per sample, (O’Sullivan et al., 2011)
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I was not able to find specific incidence of the following techniques used on sand flies, but they could be used to identify the vector, pathogen, or source of blood meal. For example, nested PCR, which uses the sequence amplified in the first PCR to do the second PCR, and Loop-mediated Isothermal Amplification (LOOP PCR) which simply uses Bst polymerase to denature the DNA rather than temperature, (Nayduch et al., 2019).
DNA sequencing methods, putting the nucleotides of a target’s DNA in order, may also be used to identify insect vectors, pathogens or sources of blood meals, but sequencing can be cost prohibitive. This method is useful when studying the host meal of a vector that the range of hosts is unknown for, (Kent, 2009).
DNA profiling (fingerprinting) can be used as well. This method creates a genetic profile based on allele sizes at numerous different loci, (Kent, 2009). This technique has been used to characterize the hosts of a vector down to the specific individuals of a species that the insect has taken a blood meal from, (Kent, 2009).
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