Systems and methods for targeting Dirofilaria immitis and Dirofilaria repens

Abstract

To provide platforms, including devices, systems, kits, and methods for the differential detection of Dirofilaria immitis and Dirofilaria repens in an animal host using parasite-specific DNA target capture techniques as well as polymerase chain reaction detection of those targets.SOLUTION: In accordance with one embodiment of the invention, provided is a device for diagnosis of a parasite infection in an animal host, the device including a PCR measurement apparatus, having a thermocycler.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 839,136, filed Apr. 26, 2019, and U.S. Provisional Patent Application No. 62 / 871,463, filed Jul. 8, 2019, each of which is hereby incorporated by reference in its entirety.

[0002] Technical Field The present invention relates to systems and methods for identifying infections by parasites in an animal host, particularly infections by Dirofilaria immitis and Dirofilaria repens.

Background Art

[0003] Canine filariasis, and to a lesser extent feline filariasis, are caused by infection with the parasitic roundworm Dirofilaria immitis [Haddock, K. C. Soc. Sci. Med. (1987); Knight, D. H. Veterinary Clinics of North America - Small Animal Practice (1987); Shearer, P. Banf. Appl. Res. Knowl. Team 1-16 (2011)]. Filariasis is one of the most important health problems for companion animals in the United States and worldwide [Wang, D. et al. Parasites and Vectors 7, 1-18 (2014); Dantas-Torres, F. and Otranto D. Parasites and Vectors 6, 1 (2013)]. This parasite is also known to infect other mammals, including wolves, coyotes, bears, foxes, seals, sea lions, and the endangered North American river otter (Lontra canadensis). D. immitis occasionally infects humans, but rarely causes a significant clinical picture [Ro, J. Y. et al. Human Pathology (1989)]. D. immitis is most prevalent in North and South America, Europe, Japan, and Australia.

[0004] Dirofilaria repens is a parasitic roundworm related to D. immitis that causes diseases in dogs, cats, wolves, coyotes, foxes, seals, and humans [Genchi, C. and Kramer, L. Parasites and Vectors 10, 1-6 (2017)]. D. repens causes various clinical manifestations in dogs, including various types of severe dermatitis, skin nodules, and in severe cases, organ damage [Capelli, G. et al. Parasites and Vectors 11, 1-21 (2018)]. D. repens is an Old World disease mainly found in Europe, Africa, and South Asia [Genchi, C. and Kramer, L. H. Vet. Parasitol. 280, 108995 (2019)]. Similar to D. immitis, D. repens is also transmitted by mosquitoes. Also, similar to D. immitis, D. repens sometimes causes infections in humans [Capelli, G. et al. Parasites and Vectors 11, 1-21 (2018); Harizanov, R. N. et al. Parasitol. Res. 113, 1571-1579 (2014)].

[0005] Ideally, all dogs and cats, both pets and strays, should be tested, preferably periodically, for the presence of D. immitis. Such testing is particularly important when the host mammal is receiving a prophylactic drug regimen. Current tests include direct observation of microfilariae in the blood, antigen tests, and polymerase chain reaction ("PCR") [Trancoso, T.A.L. et al. Rev Bras Parasitol Vet. 29, 1, (2020)]. The direct observation of microfilariae in blood using a microscope is the oldest test but suffers from lack of sensitivity because low levels of microfilariae are often missed, or because there are no microfilariae in the blood despite the presence of adult worms in the body of the host mammal (infection with only one sex, sexually immature worms, worms too old to reproduce, or due to the immune system of the host mammal that is successful in killing microfilariae but not adult worms). Furthermore, the direct microscopic evaluation of blood for mass screening of large populations of host mammals is not practical due to the time and skill level required.

[0006] Currently, the most commonly used test for screening dogs for D. immitis is an antigen test based on the detection of circulating D. immitis antigens that have flowed into the bloodstream from sexually mature female adults [Little, S. et al. Parasites and Vectors 11, 1-10 (2018); Ciuca, L. et al. Vet. Parasitol. 225, 81-85 (2016)]. There are several significant problems with antigen-based tests. First, this test does not effectively detect D. immitis parasites in mammalian hosts until the female parasite is sexually mature (6-7 months after infection). This is an important period. A delay in detection allows the parasite to get a head start, but if treatment can be started early, the time the parasite has to cause harm to the host animal is reduced, and treatment effectiveness is significantly improved. Furthermore, antigen-based tests do not effectively detect male infections in only one sex. Additionally, approximately 1% of host mammals with a negative antigen test result have been shown to be microfilaria positive (false negative results). Finally, there is considerable evidence that antigen-based assays can cross-react with other parasite species and cause false positive results [Schnyder, M. and Deplazes, P. Parasit Vectors. 5, 258 (2012); Aroch, I. et al. Vet Parasitol. 211, 303-305 92015)].

[0007] To address issues related to microscope-based and antigen-based tests, several DNA-based PCR tests have been developed [Gioia, G. et al. Vet. Parasitol. 172, 160-163 (2010); Norgen Biotek. Dirofilaria immitis PCR Detection Kit (2011); Latrofa, M. S. et al. Vet. Parasitol. 185, 181-185 (2012); Albonico, F. et al. Vet. Parasitol. 200, 128-132 (2014); Tahir, D. et al. Vet. Parasitol. 235, 1-7 (2017)]. However, all of these PCR tests are designed to detect low-copy number DNA targets found in the genomes of D. immitis and / or D. repens, and thus are not highly sensitive. Due to these tests being unable to detect very low-level or prepatent infections, i.e., infections prior to the period when D. immitis or D. repens infections can be detected using serological antigen tests, for example, infections prior to the female adult becoming sexually mature and producing microfilariae, these tests have been used conservatively or not at all in the veterinary environment.

[0008] The arguments for a highly sensitive species-specific test for D. repens are similar to those for the test for D. immitis. In D. repens, microscopy-based tests are mainly used to search for the presence of microfilariae [Capelli, G. et al. Parasites and Vectors 11, 1-21 (2018)]. There is no standardized antigen-based test specific for D. repens available for screening host mammals [Ciuca, L. et al. Vet. Parasitol. 225, 81-85 (2016)]. Therefore, there is a great need for a highly sensitive species-specific assay that can detect small amounts of DNA in the blood or serum of host mammals for the presence of D. immitis and / or D. repens at all life cycle stages. A highly sensitive species-specific PCR test for detecting D. immitis or D. repens in mosquito hosts has not been developed. Nevertheless, knowledge of the geographical distribution and density of D. immitis and D. repens in mosquito populations is an important factor when planning parasite control programs. In order to understand the degree of risk to local dog and cat populations, the geographical distribution of infected mosquitoes must be accurately evaluated. Therefore, there is a need for a highly sensitive species-specific test for detecting D. immitis and D. repens at any life cycle stage in pools of mosquitoes collected in the field.

Prior Art Documents

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Summary of the Invention

Means for Solving the Problems

[0010] According to an embodiment of the present invention, there is provided a device for diagnosing parasitic infections in an animal host, the device comprising a PCR measurement device including a thermocycler. The PCR measurement device is configured to target repetitive sequences in the DNA of the parasite in a tissue sample obtained from the infected animal host.

[0011] The parasite can be Dirofilaria immitis or Dirofilaria repens, and the repetitive sequence can be selected to provide sensitivity and selectivity to the parasite. In some embodiments, the PCR measurement device includes an optical readout mechanism, which optionally includes a test strip having a visible band.

[0012] The tissue sample can be plasma, serum or whole blood obtained from an infected animal host. Additionally, the tissue sample can be from an infected mosquito.

[0013] According to an embodiment of the present invention, a method for treating a mammalian host suspected of being infected with a parasite is provided. The method includes (a) providing a sample from a mammalian host suspected of being infected with a parasite; and (b) causing detection of the presence of the parasite in the sample. This detection includes (i) making DNA available from the sample; (ii) mixing the sample with a labeled DNA complementary to the target DNA; and (iii) detecting the target DNA using PCR. If the presence of the parasite is detected as a result of the step of causing detection, an effective treatment to reduce or eliminate parasite infection is administered to the mammalian host.

[0014] The labeled DNA is produced using a process that includes (a) a step of identifying a target DNA, the target DNA being repetitive DNA uniquely possessed by a parasite. The identifying step includes (i) obtaining genomic DNA sequence reads from the parasite, (ii) trimming each sequence read to produce a set of trimmed sequence reads of equal length, (iii) comparing each sequence in the set of reads to all other sequence reads in the set of reads to form read pairs, where each read of a given pair shares at least 90% similarity with the other read of the pair over at least 55% of its length, (iv) evaluating the read pairs to identify a candidate set of sequences in the parasite's genome that are presumed to have a high copy number in the genome, and (v) selecting a final sequence uniquely possessed by the parasite from the candidate set of sequences; and (b) causing the synthesis of the labeled DNA using the final sequence uniquely possessed by the parasite, the labeled DNA being complementary to the identified repetitive DNA uniquely possessed by the parasite.

[0015] The parasite can be D. immitis or D. repens. In some embodiments, the parasite infection is patent. In other embodiments, the parasite infection is pre-patent. The tissue sample can be plasma, serum, or whole blood obtained from an infected mammalian host that can be a dog.

[0016] In some embodiments, the labeled DNA includes a conjugation moiety. The conjugation moiety can be biotin. In some embodiments, the target DNA is isolated from the mixture using a capture medium that binds to the capture DNA. The capture medium can be magnetic beads or a test strip, and the capture medium can be coated with streptavidin.

[0017] In some embodiments, the PCR is real-time PCR using a primer and probe set. In some embodiments, the parasite is D. immitis, and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, and p3Dim1. In other embodiments, the parasite is D. repens, and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1, and p3Dre1.

[0018] In some embodiments, the parasite is D. immitis and the target DNA is Dim1. The labeled DNA can be a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

[0019] In some embodiments, the parasite is D. repens and the target DNA is Dre1. The labeled DNA can be a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

[0020] In some embodiments, the treatment is selected from the group consisting of melarsomine, ivermectin, doxycycline, moxidectin, and combinations thereof.

[0021] According to embodiments of the present invention, a kit for detecting parasite infection in an animal host is provided. The kit includes (a) labeled DNA complementary to repetitive species-specific parasite target DNA, (b) a capture medium that binds to the capture DNA, and (c) a set of written instructions for detecting parasite infection.

[0022] The parasite can be D. immitis or D. repens, and the labeled DNA includes a conjugation moiety. In some embodiments, the conjugation moiety is biotin, and the capture medium is selected from the group consisting of magnetic beads, test strips, and combinations thereof. In some embodiments, the capture medium is coated with streptavidin. The kit can include a primer and probe set.

[0023] In some embodiments, the parasite is D. immitis and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the parasite is D. immitis and the target DNA is Dim1. In some embodiments, the parasite is D. immitis and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, p3Dim1, and p4Dim1.

[0024] In other embodiments, the parasite is D. repens and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8. In some embodiments, the parasite is D. immitis and the target DNA is Dre1. In some embodiments, the parasite is D. repens and the target DNA is Dre1. In some embodiments, the parasite is D. repens and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1, and p3Dre1. In certain embodiments, for example, the following are provided: (Item 1) A device for diagnosing parasite infection in an animal host, comprising a PCR measurement device including a thermocycler, wherein the PCR measurement device is configured to target repetitive sequences in the DNA of the parasite in a tissue sample obtained from the infected animal host. (Item 2) The device according to item 1, wherein the parasite is selected from the group consisting of Dirofilaria immitis and Dirofilaria repens. (Item 3) The device according to any one of items 1 to 2, wherein the repetitive sequences are selected to provide sensitivity and selectivity for the parasite. (Item 4) The device according to any one of items 1 to 3, wherein the PCR measurement device includes an optical reading mechanism. (Item 5) The device according to item 4, wherein the accompanying optical reading mechanism includes a test strip having a visible band. (Item 6) The device according to any one of items 1 to 5, wherein the tissue sample is selected from the group consisting of plasma, serum, and whole blood obtained from the infected animal host. (Item 7) The device according to any one of items 1 to 6, wherein the tissue sample is derived from an infected mosquito. (Item 8) A method for treating a mammalian host suspected of being infected with a parasite, (a) providing a sample from the mammalian host suspected of being infected with the parasite; and (b) causing detection of the presence of the parasite in the sample, the detection comprising: (i) making DNA available from the sample; (ii) mixing the sample with a labeled DNA complementary to the target DNA; and (iii) detecting the target DNA using PCR comprising the steps comprising, wherein if the presence of the parasite is detected as a result of the step of causing detection, performing on the mammalian host a treatment effective to reduce or eliminate the parasite infection. (Item 9) The labeled DNA is a. identifying the target DNA, wherein the target DNA is repetitive DNA uniquely possessed by the parasite, and the identifying step comprises: i. obtaining genomic DNA sequence reads from the parasite, ii. trimming each sequence read to generate a set of trimmed sequence reads of equal length, iii. To form read pairs, comparing each array of the read set with all other array reads in the read set, wherein each read of a given pair shares at least 90% similarity with the other read of the pair over at least 55% of its length; iv. Evaluating the read pairs to identify a candidate set of sequences in the genome of the parasite that are presumably present at high copy numbers in the genome; and v. Selecting a final sequence uniquely held by the parasite from the candidate set of sequences Steps including; and b. Using the final sequence uniquely held by the parasite to initiate synthesis of the labeled DNA A method according to item 8, which is produced using a process including, wherein the labeled DNA is complementary to the identified repetitive DNA uniquely held by the parasite. (Item 10) The method according to any one of items 8 to 9, wherein the parasite is selected from the group consisting of D. immitis and D. repens. (Item 11) The method according to item 10, wherein the parasitic infection is patent. (Item 12) The method according to item 10, wherein the parasitic infection is pre-patent. (Item 13) The method according to any one of items 8 to 12, wherein the tissue sample is selected from the group consisting of plasma, serum, and whole blood obtained from an infected mammalian host. (Item 14) The method according to any one of items 8 to 13, wherein the mammalian host is a dog. (Item 15) The method according to any one of items 8 to 14, wherein the labeled DNA contains a conjugation moiety. (Item 16) The method according to item 15, wherein the conjugation moiety is biotin. (Item 17) The method according to any one of Items 8 to 16, wherein the target DNA is isolated from the mixture using a capture medium that binds to the capture DNA. (Item 18) The method according to Item 17, wherein the capture medium is selected from the group consisting of magnetic beads and test strips. (Item 19) The method according to any one of Items 17 to 18, wherein the capture medium is coated with streptavidin. (Item 20) The method according to any one of Items 8 to 19, wherein the PCR is real-time PCR using a primer and a probe set. (Item 21) The method according to Item 20, wherein the parasite is D. immitis and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, and p3Dim1. (Item 22) The method according to Item 20, wherein the parasite is D. repens and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1, and p3Dre1. (Item 23) The method according to any one of Items 8 to 21, wherein the parasite is D. immitis and the target DNA is Dim1. (Item 24) The method according to any one of Items 8 to 21 and 23, wherein the parasite is D. immitis and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4. (Item 25) The method according to any one of Items 8 to 20 and 22, wherein the parasite is D. repens and the target DNA is Dre1. (Item 26) The method according to any one of items 8 to 20, 22, and 25, wherein the parasite is D. repens and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8. (Item 27) The method according to any one of items 8 to 26, wherein the treatment is selected from the group consisting of melarsomine, ivermectin, doxycycline, moxidectin, and combinations thereof. (Item 28) A kit for detecting parasite infection in an animal host, comprising: d) labeled DNA complementary to repetitive species-specific parasite target DNA, e) a capture medium that binds to the capture DNA, and f) a set of written instructions for detecting the parasite infection A kit comprising: (Item 29) The kit according to item 28, wherein the parasite is selected from the group consisting of D. immitis and D. repens. (Item 30) The kit according to any one of items 28 to 29, wherein the labeled DNA comprises a conjugation moiety. (Item 31) The kit according to item 30, wherein the conjugation moiety is biotin. (Item 32) The kit according to any one of items 28 to 31, wherein the capture medium is selected from the group consisting of magnetic beads, test strips, and combinations thereof. (Item 33) The kit according to any one of items 28 to 32, wherein the capture medium is coated with streptavidin. (Item 34) The kit according to any one of items 28 to 33, comprising a primer and a probe set. A kit. (Item 35) The kit according to any one of items 28 to 34, wherein the parasite is D. immitis and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4. (Item 36) The kit according to any one of items 28 to 35, wherein the parasite is D. immitis and the target DNA is Dim1. (Item 37) The kit according to item 34, wherein the parasite is D. immitis and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, p3Dim1, and p4Dim1. (Item 38) The kit according to any one of items 28 to 34, wherein the parasite is D. repens and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8. (Item 39) The kit according to any one of items 28 to 34 and 38, wherein the parasite is D. repens and the target DNA is Dre1. (Item 40) The kit according to item 34, wherein the parasite is D. repens and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1, and p3Dre1.

[0025] The above features of the embodiments will be more easily understood with reference to the following detailed description and the accompanying drawings.

Brief Description of the Drawings

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[0032] Detailed Description of Specific Embodiments Definitions. As used in this specification and the appended claims, the following terms shall have the indicated meanings unless the context clearly requires otherwise:

[0033] "Set" includes at least one member.

[0034] As used herein, "real-time polymerase chain reaction", "real-time PCR", "quantitative polymerase chain reaction", and "qPCR" are synonymous.

[0035] "Thermocycler" is an apparatus configured to amplify segments of DNA via polymerase chain reaction (PCR) by performing a series of cycles of temperature changes that cause replication of the DNA. See, for example, Mullis KB (April 1990) Scientific American. 262 (4): 56-61, 64-5. Saiki R et al. (December 1985) Science. 230 (4732): 1350-4. See also.

[0036] The "accompanying optical reading mechanism" is a system that includes a set of DNA-binding labeled sequence-specific primers and probes, combined with a mechanism for optically reporting the presence of a threshold when an accompanying thermocycler reaches the quantification cycle Cq. The optical reading mechanism can be achieved by using fluorescently labeled sequence-specific primers and probes, as well as a photographic optical detector similar to those in standard qPCR systems available, for example, from Applied Biosystems (ThermoFisher, Waltham, Massachusetts, USA). Alternatively, the optical reading mechanism can be performed by using appropriately labeled DNA to induce the appearance of visible bands on a test strip, as described in Zaky WI et al. (2018) PLoS Negl Trop Dis 12(11): e0006962. As described, it can be carried out by using appropriately labeled DNA to induce the appearance of visible bands on a test strip.

[0037] The "configured PCR measurement device" is a PCR measurement device equipped with primers for a specific DNA target.

[0038] As used herein, "infected", e.g., an infected animal host, an infected mammalian host, or an infected mosquito host, shall mean that the organism harbors the parasite D. immitis or D. repens in its body.

[0039] "Prepatent infection", etc., shall mean a D. immitis or D. repens infection that exists before the period when the D. immitis or D. repens infection can be detected using a serological antigen test.

[0040] "Patent infection", etc., shall mean a D. immitis or D. repens infection that exists after the period when the D. immitis or D. repens infection can be detected using a serological antigen test.

[0041] The "conjugation moiety" shall mean a chemical moiety attached to a nucleic acid molecule that promotes the binding of the nucleic acid molecule to a capture medium, such as magnetic beads, non-magnetic beads, membranes, test strips (used during strip tests), or the contents of a column.

[0042] "Capture DNA" shall mean a member of the group consisting of (a) a capture oligonucleotide having a conjugation moiety, (b) DNA synthesized from a PCR primer having a conjugation moiety, and (c) combinations thereof.

[0043] "Capture medium" shall mean any medium that specifically binds to a particular conjugation moiety. For example, magnetic beads, non-magnetic beads, membranes, test strips (used during strip tests), and the contents of a column can be coated with molecules, proteins, or other reactive groups that interact with and bind to a particular conjugation moiety. A capture oligonucleotide having a conjugation moiety, or DNA synthesized from a PCR primer having a conjugation moiety, can bind to the capture medium.

[0044] "Labeled DNA" shall mean a member selected from the group consisting of (a) a capture oligonucleotide having a conjugation moiety, (b) a PCR primer having a conjugation moiety; (c) a real-time PCR probe containing one or more moieties to facilitate its detection, (d) a test strip probe containing one or more moieties to facilitate its detection, and (e) combinations thereof.

[0045] "Mammalian host" shall mean a mammal infected with a parasite.

[0046] "Mosquito host" and "mosquito vector" shall mean a mosquito infected with a parasite.

[0047] "Animal host" or "host", which is not qualified as a mammalian host or a mosquito host, includes both mammalian hosts and mosquito hosts.

[0048] "Complementary" shall mean that at least 80% of the bases of the oligonucleotide, primer or probe are capable of base pairing with the target sequence.

[0049] For both D. immitis and D. repens, infection begins when infectious larvae (stage 3 L3) are implanted into the skin of the mammalian host by the mosquito. The infectious larvae enter through the bite caused by the mosquito and burrow into the subcutaneous tissue. During the next 6 - 7 months, D. immitis larvae molt twice, migrate to the pulmonary artery and heart, and grow into fully adult parasites. Unlike D. immitis, D. repens adults remain in the subcutaneous tissue and do not migrate to the heart or pulmonary artery.

[0050] Male and female adults mate, and the female releases thousands of microfilariae (stage 1 L1 larvae) into the bloodstream daily. These microfilariae circulate in the blood until they are taken up by a mosquito while it is taking a blood meal. Once inside the mosquito, the L1 larvae molt to become L2, and then infectious L3 larvae. The L3 migrate to the mouthparts of the mosquito, where they can infect another mammalian host when the mosquito feeds again. Thus, the complete life cycle of both D. immitis and D. repens requires both a mammalian host and a mosquito host [Kotani, T. and Powers, K. G. Am. J. Vet. Res. 43, 2199 - 2206 (1982); Silaghi, C., Beck, R. et al. Parasites and Vectors 10, 1 - 13 (2017)]. In infected mammalian hosts, the adults can grow up to 10 - 12 inches in length and survive for 5 - 10 years. Microfilariae can survive in the bloodstream for 2 - 3 years before being taken up by a mosquito during a blood meal.

[0051] In mammalian hosts infected with D. immitis, visible symptoms include labored breathing, coughing, and wasting. Damage to the heart, lungs, and kidneys often occurs before symptoms are observed [Monchy, D., Levenes, H., Guegan, H., Poey, C. & Dubourdieu, D. Pulmonary dirofilariasis. Medecine tropicale: revue du Corps de sante colonial (1993)]. Often, the mammalian host ultimately dies due to congestive heart failure.

[0052] Various drugs containing melarsomine, which can kill adult worms, can be used to treat filariasis. However, such treatment is expensive because hospitalization is recommended and in many cases, enforced bed rest is required for several months. Before and after melarsomine treatment, other drugs are used and in some cases, it may be necessary to repeat the melarsomine treatment. This treatment is not without risks as the killed adult worms and microfilariae can produce blood clots that can clog the lungs and circulation. In many cases, especially in advanced cases where chronic symptoms are already apparent at the time of diagnosis, the disease remains fatal even with treatment due to the increased risk of blood clots and other complications. In such advanced cases, or if the animal cannot tolerate melarsomine treatment, surgery may be required to physically remove the adult worms from the pulmonary artery and heart. Therefore, prevention of initial infection is a much preferred alternative as it is safer and considerably less expensive. The risk of infection can be dramatically reduced by keeping the pet indoors, but this is often neither a practical nor a desirable approach. Administration of monthly prophylactic drugs such as ivermectin to at-risk animals is the most common approach for filariasis prevention. Such prophylactic treatment should only be initiated after a diagnostic test for the presence of the worms. Even when using monthly prophylactic preventives, regular testing for D. immitis is still recommended to ensure that the treatment is functioning properly and to detect any infections that may have been present but undetected before treatment was initiated.

[0053] Other treatments for D. immitis infection include pretreatment with ivermectin and doxycycline prior to the use of melarsomine to reduce lung damage caused by the worms. D. immitis infection can also be directly treated with macrocyclic lactones, such as ivermectin and moxidectin, in combination with doxycycline.

[0054] ​The clinical presentation of D. repens is different from that of D. immitis, although the drug treatment regimens, while not highly standardized, are similar.

[0055] A platform is described herein that includes parasite-specific DNA target capture techniques according to embodiments of the invention and devices, systems, kits, and methods for the differential detection of D. immitis and D. repens through the coupling of polymerase chain reaction ("PCR") detection of such targets. In some embodiments, the PCR is real-time PCR. Due to their high sensitivity and species specificity, these platforms can be used to screen mammalian host blood / plasma samples to detect infection with either D. immitis or D. repens as early as 10 weeks after infection. According to embodiments of the invention, patent and pre-patent infections can be detected by enabling the selective capture of genomic target DNA or cell-free target DNA ("cfDNA").

[0056] In some embodiments, the platforms described herein are capable of detecting pre-patent infections with D. immitis or D. repens. Platforms for detecting these pre-patent infections are not currently available. However, the ability to detect pre-patent infections is clinically useful and opens the door to new treatment strategies because infected animals can be treated early with low-dose drug therapy, experience fewer treatment side effects, and be less likely to suffer physiological damage due to the infection.

[0057] In some embodiments, the platforms described herein are intended for use by veterinarians and clinical laboratories to enable early detection of D. immitis or D. repens infections. In some embodiments, these platforms may also be used by epidemiologists to facilitate screening and analysis of mosquitoes, the insect vectors of these parasites. Mosquito screening enables mapping, evaluation of treatment strategies, evaluation of intervention programs, and assessment of the risk of community spread of D. immitis and D. repens infections.

[0058] The parasite detection platforms described herein were developed through sophisticated bioinformatics-based screening of the genomes of both D. immitis and D. repens. For each organism, the bioinformatics screening identified genomic DNA sequences that are unique to that organism and are predicted to be present in the largest copy number within the genome of that parasite. These DNA elements present optimal targets for the detection of their respective parasites because these DNA elements are unique to the originating species and are highly repetitive within the originating genome, thereby serving as species-specific and optimally sensitive detection assay targets. The greater the number of targets, the more sensitive the assay, so these repetitive DNA elements are sensitive targets for DNA detection assays. By designing oligonucleotide constructs having sequence complementarity to these assay targets, oligonucleotides capable of binding to the specific genomic target sequences can be used to capture DNA molecules containing the identified target sequences.

[0059] In some embodiments, the addition of the biotin label (conjugation moiety) to each capture molecule enables the binding of these artificial constructs to streptavidin-coated magnetic beads via biotin-streptavidin linkage. This linkage results in the generation of capture beads that can specifically bind (hybridize) to D. immitis or D. repens target DNA in blood / serum from mammalian hosts or mosquitoes. After specific binding and capture of the target DNA, the D. immitis and / or D. repens target DNA can be enriched via magnetic bead isolation and elution from the capture oligonucleotides.

[0060] In some embodiments, the capture oligonucleotide can first hybridize to the target DNA and then can be bound to a suitably treated capture medium, such as streptavidin-coated beads. In other embodiments, the capture oligonucleotide is first bound to a suitably treated capture medium and then hybridized to the target DNA.

[0061] In addition to the biotin / streptavidin interaction, various chemistries can be used to bind the capture DNA to a suitable capture medium. In some embodiments, the capture DNA can be modified with a 5'-sulfhydryl conjugation moiety that facilitates direct binding of the capture DNA to capture beads, such as Dynabeads™ M-270 Amine (ThermoFisher, Waltham, Massachusetts, USA), or other similarly coated capture media. In other embodiments, the capture oligonucleotide can be modified with a 5' or 3'-amino conjugation moiety, or the PCR primer can be labeled with a 5'-amino conjugation moiety that facilitates direct binding of the capture DNA to capture beads, such as Dynabeads™ M-270 Carboxylic Acid (ThermoFisher, Waltham, Massachusetts, USA), or other similarly coated capture media, via an amide bond.

[0062] In some embodiments, the I-Linker™ conjugation moiety can be attached to the 5' end of a capture oligonucleotide(s) or PCR primer. The I-Linker™ conjugation moiety can replace amino conjugation moiety modifications in many applications. Further, the I-Linker™ conjugation moiety expands the range of capture medium reactive groups that can be used for the attachment of capture DNA to a capture medium. For example, aldehyde-modified capture media and ketone-modified capture media can be used to bind to capture DNA having an I-Linker™ conjugation moiety.

[0063] In some embodiments, amine-modified capture DNA can be attached to a capture medium via exposed carboxylate groups or succinimidyl esters on the capture medium. In other embodiments, thiol-modified capture DNA can be attached to an aminosilane capture medium using a reagent crosslinker, such as 4-(maleimidophenyl)butyric acid N-succinimidyl ester (SMPB). In some embodiments, Acrydite™ can be used as a conjugation moiety for covalently attaching capture DNA to a capture medium via an acrylic linkage. In other embodiments, 5'-digoxigenin NHS ester can be used as a conjugation moiety for attaching capture DNA to a capture medium coated with anti-digoxigenin. In other embodiments, 5'-sulfhydryl-modified capture DNA can be used to bind to an amine-coated capture medium.

[0064] Carboxyl conjugation moieties and amino conjugation moieties are common reactive groups for attaching capture DNA to a capture medium. For example, capture media containing the following reactive groups can be utilized: -COOH (carboxylic acid), -RNH2 (primary aliphatic amine), -ArNH2 (aromatic amine), -ArCH2Cl chloromethyl (vinylbenzyl chloride), -CONH2 (amide), -CONHNH2 (hydrazide), -CHO (aldehyde), -OH (hydroxyl), -SH (thiol), and -COC (epoxy).

[0065] Other capture media capable of binding to the capture oligonucleotide, such as non-magnetic beads, such as silica beads, polystyrene beads, Sepharose™ beads and Sephadex® beads, can also be used. These beads can be isolated using centrifugation, after which the target DNA can be eluted. Further, column-based capture of the target DNA can be carried out by using a column packed with the above-described non-magnetic beads coated with the capture oligonucleotide. The column may be packed with magnetic beads coated with the capture oligonucleotide. The sample can pass through such a column, and if the target DNA is present, this binds to the beads coated with the capture oligonucleotide. The target DNA can subsequently be eluted from these beads.

[0066] As further described below, test strips can also be used as a capture medium for binding to DNA synthesized from PCR primers having a conjugation portion.

[0067] Other methods for capturing target DNA can include microfluidic lateral flow techniques that use capture oligonucleotides bound to a surface (membrane). Microarray-like slides or polystyrene microwells coated with the capture oligonucleotide can also be used to capture the target DNA.

[0068] In some embodiments, elution of the target DNA is performed by simply heating the capture beads or other capture medium, thereby breaking the interaction (hydrogen bond) between the capture oligonucleotide and the captured target molecule having a sequence complementary to the capture oligonucleotide. The supernatant containing these thermally released target molecules is then recovered to enable downstream testing by PCR.

[0069] Elution of the target DNA into the solution can also be carried out by performing 3 to 5 cycles of PCR on the bead-bound target DNA in order to amplify the target DNA. The amplified target DNA can then be subjected to detection via downstream tests by PCR. Furthermore, denaturing reagents, such as alkylating reagents that produce a pH higher than 11 in a DNA-containing solution, can also be used to disrupt the hydrogen bonds between the capture oligonucleotide and the captured target molecule having a sequence complementary to the capture oligonucleotide.

[0070] Exemplary capture oligonucleotides are illustrated in FIGS. 1A and 1B. The repetitive species-specific DNA sequences used to design these capture oligonucleotides are also illustrated in FIGS. 1A and 1B. An exemplary diagram of the target DNA capture procedure is shown in FIG. 2.

[0071] In some embodiments, after isolation of the target DNA from the sample via complementary sequence capture, magnetic pull-down, and thermal release, highly sensitive species-specific detection of D. immitis and / or D. repens can be achieved by using a PCR-based assay on the isolated DNA sample. The PCR-based assay was developed by leveraging the same highly sensitive and species-specific repetitive DNA elements that are targeted for capture. In some embodiments, for each parasite, PCR primers were designed to amplify these target DNA sequences, and a modified real-time PCR probe construct was designed to enable fluorescence detection after amplification.

[0072] Preferably, each PCR primer and probe is 12 base pairs (bp) to 40 bp in length. The primers and probes need not be completely complementary to their target sequences, but at least 80% of the bases of the oligonucleotide, primer, or probe should be capable of base pairing with the target sequence.

[0073] In some embodiments, probe design includes incorporation of a 6-FAM fluorophore, i.e., a fluorescent dye, linked to the 5’ end of the construct, a non-fluorescent quencher linked to the 3’ end of the construct, and an internal non-fluorescent quencher. Exemplary primer and probe constructs are illustrated in FIGS. 3A and 3D.

[0074] A variety of real-time PCR probes containing a fluorophore, i.e., a fluorescent dye, and one or two quenchers can be utilized.

[0075] Suitable probe fluorophores include, but are not limited to, 5’ 6-FAM, 5’ TET, 5’ Yakima Yellow®, 5’ HEX, 5’ JOE, 5’ Cy3, 5’ Texas Red-X®, 5’ Cy5, 5’ MAX, 5’ TYE 563, 5’ TAMRA, 5’ ROX, 5’ TEX 615, and 5’ TYE 665.

[0076] In various embodiments of the present invention, suitable quenchers, readily apparent to those skilled in the art, are also combined with the aforementioned probe fluorophores. These quenchers include, but are not limited to, ZEN™ (internal quencher) plus 3’ Iowa Black FQ®, 3’ Iowa Black RQ-Sp®, TAO™ (internal quencher) plus Iowa Black RQ-Sp®, and Black Hole Quencher1. See, e.g., Integrated DNA Technologies (Coralville, Iowa, USA) and Silaghi C. et al. Parasit Vectors. 10, 1, (2017). TAMRA quencher or MGB-NFQ quenching chemistry can also be used (ThermoFisher Scientific (Waltham, Massachusetts, USA)).

[0077] Various types of real-time PCR probes can be utilized. For example, 5' nuclease probes, such as PrimeTime qPCR Probes (Integrated DNA Technologies (Coralville, Iowa, USA)), can be used. Since the probe fluorophore is quenched by one or two quenchers, the 5' exonuclease probe is initially not fluorescent. However, during PCR amplification, the 5' exonuclease activity of DNA polymerase releases the quencher(s) from proximity to the probe fluorophore, enabling detection of probe fluorophore fluorescence. A higher signal-to-noise ratio can be achieved by using an internal quencher, such as ZEN™ and TAO™ quenchers, together with a 3' quencher. The internal quencher can be located at any internal position within the probe, but preferably, the internal quencher is 9 bases from the 5'-terminal probe fluorophore.

[0078] Probes utilizing modified nucleotides can also be used. For example, Affinity Plus qPCR Probes (Integrated DNA Technologies (Coralville, Iowa, USA)) incorporate several locked nucleic acid (LNA) nucleotides into the probe to provide higher structural stability and increased T m to give higher specificity to the probe. Additionally, PrimeTime LNA qPCR Probes (Integrated DNA Technologies (Coralville, Iowa, USA)) utilizing one or more LNAs can be used to provide higher structural stability for short probes, and increased T m to give higher specificity to the probe.

[0079] Molecular beacon real-time PCR probes may also be used. Unlike 5' nuclease probes, molecular beacon probes have self-complementary ends that form a hairpin structure that is quenched when not bound to their target sequence. Molecular beacon probes contain a fluorophore at one end and a quencher at the other end. The fluorescent signal is generated by linearization of the probe upon hybridization to its target sequence during real-time PCR cycling.

[0080] In its most typical form, DNA exists as a double-stranded molecule consisting of a first strand and a second strand. The sequence of each of these strands is the reverse complement of the other, and each strand base pairs with the other strand. In some embodiments, both strands of the repetitive species-specific target DNA are captured by oligonucleotides complementary to the sequence(s) present on each strand and, in some embodiments, amplified and detected using PCR. Preferably, the sequence of the capture oligonucleotide is the same as the sequence of the PCR primer to prevent false positive amplification or a decrease in real-time PCR efficiency. In other embodiments, a single strand of the repetitive species-specific target DNA is captured by an oligonucleotide complementary to the sequence present on the strand and amplified and detected using PCR. Preferably, the sequence of the capture oligonucleotide is the same as the sequence of one of the PCR primers.

[0081] In some embodiments, test samples containing potentially infected blood / serum samples from dogs and cats, or mosquito samples potentially harboring parasites, are processed to facilitate the release of target DNA from pathogens, if present. The target DNA is then captured, isolated, and detected using PCR. For example, capture of the target DNA using magnetic beads occurs only if the sample contains D. immitis or D. repens DNA, and a positive real-time PCR signal occurs only after capture of the target DNA. In some embodiments, less than 5 copies of the captured target DNA can be detected. A single D. immitis microfilaria is estimated to contain 10 million copies of the exemplary target DNA element shown in FIG. 1A. A single D. repens microfilaria is estimated to contain 30 million copies of the exemplary target DNA shown in FIG. 1B. Furthermore, the genomic target DNA that is captured, isolated, and detected is present in all life stages, enabling the detection of both pre-patent and patent infections, as well as infections of males only, females only, and mixed male and female infections.

[0082] The nucleic acid constructs described are oligonucleotides or oligonucleotide derivatives specific for D. immitis or D. repens. Exemplary capture oligonucleotides (SEQ ID NO: 4 and SEQ ID NO: 3) for D. immitis, which are complementary to the first strand (D. immitis "target sequence") (SEQ ID NO: 1) and the second strand (SEQ ID NO: 2) of each of the exemplary target DNA elements Dim1, are shown in FIG. 1A.

[0083] Exemplary capture oligonucleotides (SEQ ID NO: 8 and SEQ ID NO: 7) complementary to the first strand (SEQ ID NO: 5) (D. repens "target sequence") and the second strand (SEQ ID NO: 6) of the exemplary target DNA element Dre1 are shown in FIG. 1B. Other D. repens target sequences are also provided. For example, SEQ ID NO: 25 can be utilized according to embodiments of the present invention. In some embodiments, SEQ ID NO: 26 can be used as a target sequence. However, SEQ ID NO: 26 has some similarity to Brugia malayi DNA. The D. repens-specific target sequences SEQ ID NO: 27 and SEQ ID NO: 28 can also be utilized according to embodiments of the present invention.

[0084] Exemplary primer and probe sets p1Dim1 for D. immitis (primer SEQ ID NO: 9 and SEQ ID NO: 10, and probe SEQ ID NO: 11) are shown in FIG. 3A. Exemplary primer and probe sets p2Dim1 for D. immitis (primer SEQ ID NO: 9 and SEQ ID NO: 21, and probe SEQ ID NO: 22) are shown in FIG. 3B. Exemplary primer and probe sets p3Dim1 for D. immitis (primer SEQ ID NO: 9 and SEQ ID NO: 23, and probe SEQ ID NO: 24) are shown in FIG. 3C.

[0085] Exemplary primer and probe sets p1Dre1 for D. repens (primer SEQ ID NO: 12 and SEQ ID NO: 13, and probe SEQ ID NO: 14) are shown in FIG. 3D. Exemplary primer and probe sets p2Dre1 for D. repens (primer SEQ ID NO: 15 and SEQ ID NO: 16, and probe SEQ ID NO: 17) are shown in FIG. 3E. Exemplary primer and probe sets p3Dre1 for D. repens (primer SEQ ID NO: 18 and SEQ ID NO: 19, and probe SEQ ID NO: 20) are shown in FIG. 3F.

[0086] In some embodiments, the D. immitis capture oligonucleotides and primer and probe sets p1Dim1, p2Dim1, p3Dim1, and p4Dim1 were designed based on output sequence data obtained from next-generation sequencing (NGS)-based analysis of adult male and female D. immitis genomic DNA (see the following Materials and Methods).

[0087] In some embodiments, the D. repens capture oligonucleotides and primer and probe sets p1Dre1, p2Dre1, and p3Dre1 were designed based on output sequence data obtained from NGS-based analysis of adult male and female D. repens genomic DNA (see the following Materials and Methods).

[0088] In some embodiments, a set of capture oligonucleotides (one capture oligonucleotide for each strand of the double-stranded target DNA) is provided for the most abundant and species-specific genomic target DNA elements of D. immitis, which is effective for isolating D. immitis-derived target DNA from samples originating from mammalian hosts or mosquito vector species and enriching the resulting samples for this target DNA. For example, the exemplary biotinylated capture oligonucleotide SEQ ID NO: 3 and SEQ ID NO: 4, or derivatives thereof, can be used to capture both strands of the target DNA Dim1. In some embodiments, a single capture oligonucleotide for capturing a single strand of the target DNA can be used. For example, one exemplary biotinylated capture oligonucleotide selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4, or derivatives thereof, can be used to capture one strand of the target DNA Dim1.

[0089] In some embodiments, a set of capture oligonucleotides (one capture oligonucleotide for each strand of the double-stranded target DNA) is provided for the most abundant and species-specific genomic target DNA element of D. repens, which is effective for isolating D. repens-derived target DNA from samples originating from mammalian hosts or mosquito vector species and enriching the obtained samples for this target DNA. For example, exemplary biotinylated capture oligonucleotide SEQ ID NO: 7 and SEQ ID NO: 8, or derivatives thereof, can be used to capture both strands of the target DNA Dre1. In some embodiments, a single capture oligonucleotide for capturing a single strand of the target DNA can be used. For example, one exemplary biotinylated capture oligonucleotide selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, or derivatives thereof, can be used to capture one strand of the target DNA Dre1.

[0090] In some embodiments, an optimal primer and probe set effective in a real-time PCR assay for detecting the presence of D. immitis target DNA in samples derived from mammalian hosts or mosquito vector species is provided. Detection of the target DNA indicates that the mammalian host or mosquito vector species is infected with D. immitis. For example, in some embodiments, an exemplary primer and probe set p1Dim1 comprising primer SEQ ID NO: 9 and SEQ ID NO: 10, or derivatives thereof, and probe SEQ ID NO: 11, or a derivative thereof, can be used. In other embodiments, an exemplary primer and probe set p2Dim1 comprising primer SEQ ID NO: 9 and SEQ ID NO: 21, or derivatives thereof, and probe SEQ ID NO: 22, or a derivative thereof, can be used. In some embodiments, an exemplary primer and probe set p3Dim1 comprising primer SEQ ID NO: 9 and SEQ ID NO: 23, or derivatives thereof, and probe SEQ ID NO: 24, or a derivative thereof, can be used.

[0091] In some embodiments, an optimal primer and probe set effective in a real-time PCR assay for detecting the presence of D. repens target DNA in a sample derived from a mammalian host or a mosquito vector species is provided. Detection of the target DNA indicates that the mammalian host or the mosquito vector species is infected with D. repens. For example, in some embodiments, an exemplary primer and probe set p1Dre1 comprising primer SEQ ID NO: 12 and SEQ ID NO: 13, or derivatives thereof, and probe SEQ ID NO: 14, or a derivative thereof, can be used. In other embodiments, an exemplary primer and probe set p2Dre1 comprising primer SEQ ID NO: 15 and SEQ ID NO: 16, or derivatives thereof, and probe SEQ ID NO: 17, or a derivative thereof, can be used. In some embodiments, an exemplary primer and probe set p3Dre1 comprising primer SEQ ID NO: 18 and SEQ ID NO: 19, or derivatives thereof, and probe SEQ ID NO: 20, or a derivative thereof, can be used.

[0092] In some embodiments, a method for treating a mammalian host suspected of being infected with a parasite is provided. The method includes (a) providing a sample from the mammalian host suspected of being infected with the parasite; and (b) causing detection of the presence of the parasite in the sample. This detection includes (i) making DNA available from the sample; (ii) mixing the sample with a labeled DNA complementary to the target DNA; and (iii) using PCR to detect the target DNA; and if the presence of the parasite is detected as a result of the step of causing detection, administering to the mammalian host a treatment effective to reduce or eliminate parasite infection.

[0093] The labeled DNA is produced using a process that includes the step of (a) identifying a target DNA, where the target DNA is repetitive DNA uniquely possessed by a parasite. This step of identification includes (i) obtaining genomic DNA sequence reads from the parasite, (ii) trimming each sequence read to produce a set of trimmed sequence reads of equal length, (iii) comparing each sequence in the set of reads to all other sequence reads in the set of reads to form read pairs, where each read of a given pair shares at least 90% similarity with the other read of the pair over at least 55% of its length, (iv) evaluating the read pairs to identify a candidate set of sequences in the parasite's genome that are presumed to have a high copy number in the genome, and (v) selecting a final sequence uniquely possessed by the parasite from the candidate set of sequences; and (b) causing the synthesis of the labeled DNA using the final sequence uniquely possessed by the parasite, where the labeled DNA is complementary to the identified repetitive DNA uniquely possessed by the parasite.

[0094] The parasite can be D. immitis or D. repens. In some embodiments, the parasite infection is patent. In other embodiments, the parasite infection is pre-patent. The tissue sample can be plasma, serum, or whole blood obtained from an infected mammalian host that can be a dog.

[0095] In some embodiments, the labeled DNA includes a conjugation moiety. The conjugation moiety can be biotin. In some embodiments, the target DNA is isolated from the mixture using a capture medium that binds to the capture DNA. The capture medium can be magnetic beads or a test strip, and the capture medium can be coated with streptavidin.

[0096] In some embodiments, the PCR is real-time PCR using a primer and probe set. In some embodiments, the parasite is D. immitis, and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, and p3Dim1. In other embodiments, the parasite is D. repens, and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1, and p3Dre1.

[0097] In some embodiments, the parasite is D. immitis and the target DNA is Dim1. The labeled DNA can be a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4.

[0098] In some embodiments, the parasite is D. repens and the target DNA is Dre1. The labeled DNA can be a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

[0099] In some embodiments, the treatment is selected from the group consisting of melarsomine, ivermectin, doxycycline, moxidectin, and combinations thereof.

[0100] In some embodiments, a kit is provided for detecting parasite infection in an animal host. The kit includes (a) labeled DNA complementary to repetitive species-specific parasite target DNA, (b) a capture medium that binds to the capture DNA, and (c) a set of written instructions for detecting parasite infection.

[0101] The parasite can be D. immitis or D. repens, and the labeled DNA includes a conjugation moiety. In some embodiments, the conjugation moiety is biotin, and the capture medium is selected from the group consisting of magnetic beads, test strips, and combinations thereof. In some embodiments, the capture medium is coated with streptavidin. The kit can include a primer and probe set.

[0102] In some embodiments, the parasite is D. immitis and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the parasite is D. immitis and the target DNA is Dim1. In some embodiments, the parasite is D. immitis and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, p3Dim1 and p4Dim1.

[0103] In other embodiments, the parasite is D. repens and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8. In some embodiments, the parasite is D. immitis and the target DNA is Dre1. In some embodiments, the parasite is D. repens and the target DNA is Dre1. In some embodiments, the parasite is D. repens and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1 and p3Dre1.

[0104] In some embodiments, there is provided a device for diagnosing D. immitis or D. repens infection in an animal host, the device comprising a PCR measurement apparatus including a thermocycler, the PCR measurement apparatus being configured to target repetitive target sequences in the DNA of D. immitis or D. repens in a tissue sample obtained from an infected animal host. The repetitive target sequences can be selected to provide sensitivity and selectivity for D. immitis or D. repens. The PCR measurement apparatus can include an optical reading mechanism. The optical reading mechanism optionally includes a test strip having a visible band. The tissue sample can be from an infected animal host. The tissue sample can be from an infected mosquito or an infected dog.

[0105] In some embodiments, a PCR-based platform utilizing test strips is provided for the detection of parasitic infections in an animal host, such as D. immitis or D. repens infections. See generally Zaky et al. PLoS Negl Trop Dis. 2018 Nov 21;12(11). This strip test platform utilizes a test strip as a capture medium. The test strip has a band coated with a reactive group for binding to DNA synthesized via PCR using PCR having a conjugation portion.

[0106] Briefly stated, tissue samples from mammals or mosquito pools suspected of being infected with D. immitis or D. repens are subjected to PCR using primers specific for target DNA, e.g., Dim1 or Dre1 respectively. One or both of the PCR primers are labeled with a conjugation moiety capable of binding to a band on a test strip. For example, as shown in FIG. 5, the reverse primer SEQ ID NO: 29 is labeled with a biotin conjugation moiety. After PCR amplification of the target DNA Dim1 using this reverse primer, the strand of DNA synthesized from SEQ ID NO: 29 during PCR is biotin-labeled and this DNA strand can then be bound to a test strip having a band coated with streptavidin. After binding of the PCR product to the test strip band, the test strip probe(s) is then hybridized to a complementary sequence on the band-bound DNA strand. The test strip probe is labeled with a moiety to facilitate its detection. For example, in FIG. 5, the test strip probe SEQ ID NO: 30 is labeled with 6-FAM. In some embodiments, the presence of this hybridized probe can be visualized using an anti-FITC antibody conjugated to gold particles (the anti-FITC antibody specifically binds to 6-FAM). Thus, if target parasite DNA is present in the tissue sample, the DNA strand synthesized during PCR of the target DNA from a PCR primer having a conjugation moiety binds to the test strip band; when bound to the test strip band, the test strip probe hybridizes to a complementary sequence on the band-bound DNA; when the test strip probe hybridizes to the band-bound DNA, its presence can be visualized, e.g., via use of an antibody that binds to the label of the test strip probe. If target parasite DNA is not present in the tissue sample, there is no DNA binding to the test strip band and visualization does not occur.

[0107] As discussed above, various conjugation moieties and capture medium chemistries other than biotin / streptavidin can be used.

[0108] Furthermore, the test strip probe may include a detectable label other than 6-FAM. Suitable labels include, but are not limited to, 5’TET, 5’Yakima Yellow®, 5’HEX, 5’JOE, 5’Cy3, 5’Texas Red-X®, 5’Cy5, 5’MAX, 5’TYE 563, 5’TAMRA, 5’ROX, 5’TEX 615, and 5’TYE 665.

[0109] Furthermore, the presence of the test strip probe can be visualized in ways other than through the use of a gold particle-conjugated antibody that specifically binds to the label of the test strip probe. For example, an alkaline phosphatase-conjugated antibody that specifically binds to the label of the test strip probe can be used with a colorimetric peroxidase substrate for visualization.

[0110] In some embodiments, the capture oligonucleotide can be used to capture target DNA from a tissue sample prior to PCR of the target DNA for strip test detection of the presence of the target DNA. In other embodiments, the capture oligonucleotide is not used prior to PCR of the target DNA for strip test detection of the presence of the target DNA.

[0111] In some embodiments, provided is a kit suitable for detecting D. immitis infection in an animal host, the kit including a set of test strip PCR primers, at least one of which has a conjugation moiety, a test strip probe (each primer and probe being complementary to a repetitive species-specific D. immitis target DNA), a capture medium that binds to DNA synthesized from the PCR primer having the conjugation moiety, and a set of written instructions for detecting D. immitis infection. At least one PCR primer of the set can be biotinylated and the capture medium can be coated with streptavidin. The capture medium can include a test strip having a band coated with a reactive group that binds to the conjugation moiety.

[0112] In some embodiments, there is provided a kit suitable for detecting D. repens infection in an animal host, the kit comprising a set of test strip PCR primers, at least one of which has a conjugation moiety, a test strip probe (each primer and probe being complementary to repetitive species-specific D. repens target DNA), a capture medium that binds to DNA synthesized from the PCR primer having the conjugation moiety, and a set of written instructions for detecting D. repens infection. At least one of the PCR primers of the set can be biotinylated and the capture medium can be coated with streptavidin. The capture medium can include a test strip having a band coated with a reactive group that binds to the conjugation moiety.

[0113] Materials and Methods The following exemplary materials and methods can be used in accordance with embodiments of the present invention. Those skilled in the art will understand that the following materials and methods are merely exemplary and that these protocols can be adjusted as needed to provide for particular circumstances.

[0114] Assay Design

[0115] The assay design described herein involves using next-generation sequencing (NGS) of a parasite genome, such as the genome of D. immitis or D. repens, to identify the most highly repeated DNA elements found in the genome of the parasite species in question, and subsequent use of a bioinformatics pipeline. This pipeline is then used to filter data on the highly repeated DNA elements in order to identify those having a DNA sequence specific to the parasite species in question. The inventors then use these highly repeated species-specific DNA sequences to design optimal capture oligonucleotides for genomic parasite target DNA containing repetitive species-specific sequences, as well as optimal PCR primers and probe sets for detecting the captured target DNA, using a bioinformatics pipeline.

[0116] Target DNA sequence identification

[0117] Paired-end next-generation sequencing-based analysis of DNA extracted from adult parasites is performed using an Illumina MiSeq® platform [300 cycle cartridge (2×150)]. In some embodiments, other NGS sequencing platforms known to those of skill in the art may also be used. After analysis, the raw reads are trimmed and filtered so that all reads are of equal length (reads less than 100 bases in length are removed and all reads greater than 100 bases are trimmed to be less than 100 bases in length). The paired-end reads are then interleaved and a subset of the reads (between 500,000 and 1,000,000) is analyzed using Galaxy-based / RepeatExplorer and TAREAN software tools (Novak P, et al. Bioinformatics. 29, 6, 2013;Novak P, et al. Nucleic Acids Res. 45, 12, 2017). These programs Enable all-against-all BLAST analysis, during which each read is compared to all other reads in the dataset. If any two reads share a similarity of 90% or higher over 55% or more of the read length, a pair is formed, and then the read pair is used to construct a cluster of reads that meet these criteria. When constructing a cluster, each read is represented by a node, and successful pair formation is indicated by the presence of an edge connecting two nodes. The length of the edge is a characteristic of the similarity between the two reads, with shorter edges connecting two reads with greater identity and longer edges indicating two reads that still meet the pair formation criteria but are less similar. Then, based on the edge length and the relatedness of the individual nodes, the cluster takes on a characteristic shape. Thus, shorter repeats form more compact star-burst-like clusters (Grant JR et al. Front Genet. 10, 833, 2019) because the individual reads within the cluster meet the pair formation criteria with more other reads in the cluster. In addition to the visual observation of the cluster structure, connectivity can be further evaluated by examining the connected component index, which is the ratio of the number of pairs within the cluster compared to the maximum number of possible pairs. For example, if all reads within a cluster meet the pair formation criteria with all other reads in the cluster, the connected component index is 1.00. Thus, a larger connected component index indicates a higher degree of sequence conservation among the reads in the cluster. This number is then used in combination with the total number of read mappings to the cluster to select candidate repeats for potential use as genomic parasite target DNA, as these two factors enable the selection of genomic repeat elements with presumably the maximum copy number. Superclusters can also be constructed when two or more clusters share a high degree of sequence similarity based on the presence of complementary paired-end read mates that are split between the two clusters.

[0118] After selection of the candidate target repetitive DNA sequences, NCBI-based BLAST analysis is performed to screen for and eliminate potential target repetitive DNA sequences that are predicted to have significant similarity to DNA from other related organisms (e.g., host species, related parasitic species, symbiotic bacteria found in the host, etc.). Through such screening, the likelihood of selecting parasitic target DNA that exhibits cross-reactivity with other species that could interfere with the specificity of the assay is greatly reduced. Next, PrimerQuest software is used to select possible primer / probe candidates to provide optimal PCR amplification of the target repetitive DNA element. These candidates are further screened using NCBI-based Primer-BLAST analysis to further eliminate primer pairs that could give rise to related off-target PCR amplification.

[0119] Capture oligonucleotide design

[0120] A pair of 5'-biotinylated capture oligonucleotides (SEQ ID NO: 3 and SEQ ID NO: 4) was designed to be complementary to each strand of double-stranded D. immitis target DNA (Dim1) (Figure 1A). This enables capture of both strands of the D. immitis target DNA.

[0121] Similarly, a pair of 5'-biotinylated capture oligonucleotides (SEQ ID NO: 7 and SEQ ID NO: 8) was designed to be complementary to each strand of double-stranded D. repens target DNA (Dre1) (Figure 1B). This enables capture of both strands of the D. repens target DNA.

[0122] Isolation of canine plasma from collected whole blood samples

[0123] Collect whole blood in a Cell-Free DNA BCT® Blood Collection Tube (Streck, USA) and maintain at room temperature until DNA isolation is initiated. Centrifuge the whole blood at 2,000 × g for 20 minutes to isolate plasma. Then, centrifuge the recovered plasma at 16,000 × g for 10 minutes to pellet remaining cells and debris. Transfer the supernatant to cryotubes and store at -80 °C until processing.

[0124] Capture of Parasite cfDNA from Canine Plasma Samples

[0125] The input sample consists of 200 μl plasma + 100 μl 1× binding buffer (5 mM Tris-HCl (pH 7.5); 0.5 mM EDTA; 1 M NaCl), to which 1.0 picomole of each biotinylated capture oligonucleotide is added, and the sample is heated at 95 °C for 10 minutes to denature cfDNA in the plasma sample. This step creates single-stranded cfDNA molecules and exposes complementary hybridization sites. Hybridization of the biotinylated capture oligonucleotide to the complementary cfDNA target occurs at 55 °C in a shaking incubator (750+ rpm). Bead capture of cfDNA is achieved by adding 50 μl of streptavidin-coated magnetic beads (Dynabeads™ M-270 Streptavidin, ThermoFisher) and incubating the sample at room temperature for 30 minutes in a shaking incubator (900+ rpm). This step binds cfDNA hybridized to the biotinylated capture oligonucleotide to the streptavidin-coated beads by strong streptavidin-biotin linkage. A magnet is used to isolate the beads carrying the captured parasite cfDNA. The beads are washed twice with 1× binding buffer. Elution of the captured parasite cfDNA is achieved by washing the beads in 1× SSC and then resuspending the beads in 50 μl ddH2O and incubating at 95 °C for 5 minutes. This step elutes non-biotinylated parasite cfDNA (target DNA) into water. A magnet is used to isolate the beads and recover the eluted cfDNA in the solution. 50 μl of the sample is recovered (Figure 2).

[0126] Alternatively, the target DNA can be eluted from the beads using PCR amplification. Using this method, the beads are resuspended in 50 μl of PCR mix (a PCR reaction mix with a non-biotinylated parasite-specific PCR primer pair) and directly amplified (3 - 5 cycles). Here, the supernatant contains the amplified non-biotinylated parasite target DNA. A magnet is used to isolate the beads and recover the amplified target DNA from the solution.

[0127] Mosquito Pool Sample Preparation: Crude Extraction Technique

[0128] Briefly, in a 1.7 ml microcentrifuge tube, each mosquito pool (up to 25 mosquitoes) is homogenized with 180 μl of 0.2 N NaOH for 3 minutes using a sterile plastic micro-pestle (Axygen Scientific, Union City, CA). The micro-pestle is then rinsed with an additional 180 μl of 0.2 N NaOH into the same 1.7 ml tube containing the homogenized mosquitoes, and it is confirmed that all mosquito debris has been removed from the pestle. A new clean sterile micro-pestle is used for each pool. Each tube is incubated at 75 °C for 10 minutes. 115.2 μl of 1 M Tris (pH = 8.0) and 364.8 μl of nuclease-free water are added to each sample, and the tube is thoroughly mixed for 10 seconds using a vortex mixer. The samples are centrifuged at 10,000 × g for 3 minutes, and the supernatant containing the extracted DNA is collected and transferred into a clean 1.7 ml microcentrifuge tube. Zaky et al. PLoS Negl Trop Dis. 2018 Nov 21;12(11) See

[0129] Mosquito Pool Sample Preparation: Column-Based Extraction Technique

[0130] Pools of up to 25 mosquitoes are placed in 2.0 ml microcentrifuge tubes. One or more zinc ball bearings (or similar items) are placed in each tube. 180 μl of phosphate-buffered saline (pH 7.2) is added to each tube. The tubes are vortexed on a horizontal shaking platform for 30 minutes. Each tube is briefly centrifuged to collect debris at the bottom of the tube. 20 μl of proteinase K is added to each sample. Then, 200 μl of a buffer containing chaotropic salts (e.g., Qiagen Buffer AL) is added to each sample and mixed by vortexing for 3 seconds. The tubes are then incubated at 70 °C for 10 minutes. Then, an additional 20 μl of proteinase K is added to each sample and vortexed for 3 seconds Mix by swirling. Incubate the tubes at 56 °C for 1 hour and then centrifuge at 16,000 × g or greater for 5 minutes to pellet debris. Transfer the supernatant from each tube to a new tube and add an additional 200 μl of 98% ethanol to each sample. Then apply each sample to an ion exchange column or bead-based solution (e.g., Qiagen DNeasy spin column) and centrifuge at 8,000 × g for 1 minute. Discard the flow-through, add 500 μl of ethanol-containing wash buffer (e.g., Qiagen buffer AW1) to each sample, and centrifuge at 16,000 × g or faster for 3 minutes. Repeat this ethanol wash two more times. After the final centrifugation, transfer the column to a new clean microcentrifuge tube and add 25 μl or more of elution solution (e.g., nuclease-free water, phosphate-buffered saline, Tris-EDTA, etc.) to each column. Place the elution solution on the column for at least 2 minutes. Centrifuge each column at 10,000 × g for 2 minutes to elute the DNA-containing sample from the column. Fischer, P, see et al. Ann Trop Med Parasitol. 96: 809-821 (2002).

[0131] Detection of target DNA (D. immitis)

[0132] Three real-time PCR primer and probe sets (p1Dim1, p2Dim1, and p3Dim1) (Integrated DNA Technologies, Coralville, Iowa, USA) were designed to be complementary to the D. immitis Dim1 repetitive target DNA.

[0133] For p1Dim1, the forward primer (SEQ ID NO: 9) and the probe (SEQ ID NO: 11) are complementary to the sequence (SEQ ID NO: 2) on one DNA strand of the D. immitis repetitive Dim1 target DNA. The reverse primer (SEQ ID NO: 10) is complementary to the sequence (SEQ ID NO: 1) on the opposite DNA strand of the Dim1 repetitive target DNA (Figure 3A). The probe has the fluorescent dye 6-FAM attached to its 5'-end and the quencher IABkFQ attached to its 3'-end. Further, the probe has an internal ZEN (trademark) quencher of 9 bases from the 5'-end. The real-time PCR example in this specification uses the primer and probe set p1Dim1.

[0134] For p2Dim1, the forward primer (SEQ ID NO: 9) and the probe (SEQ ID NO: 22) are complementary to the sequence (SEQ ID NO: 2) on one DNA strand of the D. immitis repetitive Dim1 target DNA. The reverse primer (SEQ ID NO: 21) is complementary to the sequence (SEQ ID NO: 1) on the opposite DNA strand of the Dim1 repetitive target DNA (Figure 3B). The probe has the fluorescent dye 6-FAM attached to its 5'-end and the quencher IABkFQ attached to its 3'-end. Further, the probe has an internal ZEN (trademark) quencher of 9 bases from the 5'-end.

[0135] For p3Dim1, the forward primer (SEQ ID NO: 9) and the probe (SEQ ID NO: 24) are complementary to the sequence (SEQ ID NO: 2) on one DNA strand of the D. immitis repetitive Dim1 target DNA. The reverse primer (SEQ ID NO: 23) is complementary to the sequence (SEQ ID NO: 1) on the opposite DNA strand of the Dim1 repetitive target DNA (Figure 3C). The probe has the fluorescent dye 6-FAM attached to its 5'-end and the quencher IABkFQ attached to its 3'-end. Further, the probe has an internal ZEN (trademark) quencher of 9 bases from the 5'-end.

[0136] Thermal cycling was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). A 20 μl real-time PCR reaction was set up using the following reagents: 10 μl of 2× TaqPath ProAmp Master Mix (ThermoFisher Scientific, Waltham, Massachusetts); 0.8 μl of 20 μM forward primer; 0.8 μl of 20 μM reverse primer; 2.5 μl of 1 μM probe; 0.9 μl of ddH2O; and 5 μl of template DNA (e.g., eluted Dim1 target DNA). The real-time PCR cycling conditions were as follows: 2 minutes at 50 °C and then 10 minutes at 95 °C, followed by 40 cycles of 15 seconds at 95 °C and 1 minute at 62 °C.

[0137] Detection of target DNA (D. repens)

[0138] Three real-time PCR primer and probe sets (p1Dre1, p2Dre1 and p3Dre1) (Integrated DNA Technologies, Coralville, Iowa, USA) were designed to be complementary to the D. repens Dre1 repetitive target DNA.

[0139] For p1Dre1, the forward primer (SEQ ID NO: 12) and probe (SEQ ID NO: 14) are complementary to a sequence (SEQ ID NO: 6) on one DNA strand of the D. repens repetitive Dre1 target DNA. The reverse primer (SEQ ID NO: 13) is complementary to a sequence (SEQ ID NO: 5) on the opposite DNA strand of the Dre1 repetitive target DNA (Figure 3D). The probe has a fluorescent dye 6-FAM attached to its 5' end and a quencher IABkFQ attached to its 3' end. Further, the probe has an internal ZEN™ quencher of 9 bases from the 5' end.

[0140] For p2Dre1, the forward primer (SEQ ID NO: 15) and the probe (SEQ ID NO: 17) are complementary to the sequence (SEQ ID NO: 6) on one DNA strand of the D. repens repetitive Dre1 target DNA. The reverse primer (SEQ ID NO: 16) is complementary to the sequence (SEQ ID NO: 5) on the opposite DNA strand of the Dre1 repetitive target DNA (Figure 3E). The probe has a fluorescent dye 6-FAM attached to its 5'-end and a quencher IABkFQ attached to its 3'-end. Further, the probe has an internal ZEN™ quencher of 9 bases from the 5'-end.

[0141] For p3Dre1, the forward primer (SEQ ID NO: 18) and the probe (SEQ ID NO: 20) are complementary to the sequence (SEQ ID NO: 6) on one DNA strand of the D. repens repetitive Dre1 target DNA. The reverse primer (SEQ ID NO: 19) is complementary to the sequence (SEQ ID NO: 5) on the opposite DNA strand of the Dre1 repetitive target DNA (Figure 3F). The probe has a fluorescent dye 6-FAM attached to its 5'-end and a quencher IABkFQ attached to its 3'-end. Further, the probe has an internal ZEN™ quencher of 9 bases from the 5'-end.

[0142] Thermal cycling is performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). For each of the three primer and probe sets (p1Dre1, p2Dre1, and p3Dre1), a 20 μl real-time PCR reaction is set up using the following reagents: 10 μl of 2× TaqPath ProAmp Master Mix; 0.8 μl of 20 μM forward primer; 0.8 μl of 20 μM reverse primer; 2.5 μl of 1 μM probe; 0.9 μl of ddH2O; and 5 μl of template DNA (e.g., eluted Dre1 target DNA). The real-time PCR cycling conditions are as follows: 2 minutes at 50 °C and then 10 minutes at 95 °C, followed by 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C.

[0143] DNA Capture / Isolation for Strip Assay

[0144] Total cfDNA was isolated from canine plasma using the Thermofisher MagMAX™ Cell-Free DNA Isolation Kit according to the manufacturer's instructions. For each canine sample, 2 ml of plasma was used as the starting material and the elution volume was 30 μl. 5 μl of the eluate was used as the template for each PCR reaction (35 cycles) using primer SEQ ID NO: 9 and SEQ ID NO: 29.

[0145] Alternatively, parasite-specific cfDNA can be captured using a capture medium, e.g., magnetic beads surface appropriately coated with capture oligonucleotides that bind to the capture oligonucleotides. The oligonucleotides can first be bound to the appropriately treated capture medium and then hybridized to the target DNA, e.g., Dim1. The target DNA can be eluted from the beads using PCR amplification (35 cycles) using strip test PCR primers (SEQ ID NO: 9 and SEQ ID NO: 29).

[0146] PCR Amplification for Strip Assay

[0147] Thermal cycling was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). Each PCR reaction was set up as follows: 25 μl of 2× TaqPath ProAmp Master Mix; 1 μl of 10 μM forward primer (SEQ ID NO: 9); 1 μl of 10 μM biotinylated reverse primer (SEQ ID NO: 29); 5 μl of eluate (template); and 18 μl of ddH2O. The PCR cycling conditions were as follows: 10 minutes at 95°C, followed by 35 cycles of 40 seconds at 95°C and 1 minute at 60°C, and a single 10-minute extension at 60°C.

[0148] Detection for strip assay

[0149] Combine 10 μl of the PCR product with 1 μl of a 10 pmol / μl strip test probe (SEQ ID NO: 30) and 39 μl of annealing buffer (10 mM Tris, pH 7.5 - 8, 50 mM NaCl, and 1 mM EDTA). The PCR product is then denatured by heating the mixture at 95°C for 5 minutes. The mixture is then maintained at 55°C for 30 minutes to hybridize the strip test probe to the biotinylated DNA strand of the PCR product.

[0150] Expose the test strip to the mixture next, and via lateral flow, the mixture migrated across the full length of the test strip. A visible positive result occurs when the biotinylated strand hybridized to the 6-FAM labeled probe binds to a band on the streptavidin-coated test strip (capture medium). The gold-labeled anti-fluorescein (6-FAM) antibody that binds to the 6-FAM labeled probe enables visualization of the Dim1 PCR product on the test strip. See Zaky et al. PLoS Negl Trop Dis. 2018 Nov 21;12(11).

Example

[0151] (Example 1) Analytical sensitivity of the Dim1 D. immitis real-time PCR assay The analytical sensitivity of the Dim1 D. immitis real-time PCR assay was first evaluated using the p1Dim1 primer and probe set with serial diluted amounts (100 picograms, 10 picograms, 1 picogram, 100 femtograms, 10 femtograms, 1 femtogram, 100 attograms, 10 attograms, and 1 attogram) of D. immitis genomic DNA as templates. All reactions were performed in quintuplicate. Real-time amplification of D. immitis DNA was indicated by the cycle number at which probe fluorescence was detected at the threshold, as indicated by the cycle quantification (Cq) value. If amplification occurred with an average Cq value of less than 40, the result was considered positive. The mean Cq and standard deviation (SD) of the five replicates for each input amount of genomic DNA are shown in Table 1. The D. immitis real-time PCR assay consistently detected D. immitis genomic DNA in all replicates down to an input of 10 attograms of D. immitis genomic DNA. However, only 2 out of 5 replicates showed D. immitis DNA detection in the 1 attogram input template DNA. Based on these data, the analytical sensitivity of the D. immitis Dim1 real-time PCR assay was determined to be between 10 attograms and 1 attogram of D. immitis genomic DNA.

[0152] [Table 1]

[0153] (Example 2) Species specificity of the Dim1 D. immitis real-time PCR assay The species specificity of the Dim1 D. immitis real-time PCR assay was demonstrated using genomic DNA from various related filarial parasites, along with canine and feline genomic DNA. 100 pg of input genomic DNA from the following parasites was used as a template: Brugia malayi, Loa loa, Brugia pahangi, Acanthocheilonema viteae, Wuchereria bancrofti, Onchocerca volvulus, and Onchocerca ochengi. Canine and feline genomic DNA (10 nanograms, 1 nanogram, and 100 picograms) was used as a template to test for cross-reactivity with genomic DNA from these mammalian host species. No signal was detected in any of the samples tested, demonstrating the absence of cross-reactivity of the Dim1 assay with various available parasite, canine, and feline DNA samples (Table 2).

[0154]

Table 2

[0155] (Example 3) Comparison of the sensitivity between the Dim1 real-time PCR assay and two current PCR assays Between the inventors' D. immitis assay (Dim1 real-time PCR assay) and two current real-time PCR assays, one targeting the D. immitis mitochondrial cytochrome C oxidase subunit 1 ("mitochondrial COI") gene (Tahir et. al. Veterinary Parasitology 235, 1-7 (2017)) and the other targeting the D. immitis 16S ribosomal RNA ("16S rRNA") gene (Watts et. al. Molecular and Cellular Probes 14, 425-430 (1999)) The comparison of amplification efficiency was carried out using D. immitis genomic DNA as a template. Each sample was tested in triplicate. The average Cq values obtained demonstrated that our Dim1 real-time PCR assay detected D. immitis genomic DNA 5.6 cycles earlier (using 10 pg of input DNA) and 6.5 cycles earlier (using 1 pg of input DNA) during amplification compared to the mitochondrial COI assay (Table 3). Similarly, the Dim1 real-time PCR assay detected D. immitis genomic DNA approximately 4 cycles earlier across all amounts of input DNA (100 pg, 10 pg, and 1 pg) compared to the 16S rRNA assay (Table 4). These data show an increase in the sensitivity of the D. immitis Dim1 real-time PCR assay compared to both the mitochondrial COI and 16S rRNA real-time PCR assays. This increase in sensitivity was approximately 65-fold compared to the mitochondrial COI assay and approximately 16-fold compared to the 16S rRNA assay.

[0156] [Table 3]

[0157] [Table 4]

[0158] (Example 4) Comparison of the detection limits between the Dim1 real-time PCR assay and two currently used assays The inventors compared the analytical detection limits of their Dim1 real-time PCR assay with those of the currently used 16S rRNA assay and the currently used mitochondrial COI assay using serially diluted D. immitis genomic DNA (10 picograms, 1 picogram, 100 femtograms, 10 femtograms, 1 femtogram, 100 attograms, 10 attograms, 1 attogram, and 100 zeptograms) as templates. Consistent detection (indicated by "X" in Table 5) was determined by Cq values below 40 in at least two of three replicate reactions. As shown in Table 5, the inventors' Dim1 real-time PCR assay showed consistent detection up to 10 ag of D. immitis genomic DNA, while the 16S rRNA assay and the mitochondrial COI assay showed consistent detection up to 1 fg of D. immitis genomic DNA. This data indicates that the analytical sensitivity of Dim1 real-time PCR assay detection is approximately 100-fold higher than that of the currently used real-time PCR assays. Therefore, the D. immitis Dim1 real-time PCR assay can provide increased detection sensitivity for prepatent infections in canine and feline patient samples.

[0159]

Table 5

[0160] (Example 5) Evaluation of the Dim1 Real-Time PCR Assay for Canine Blood Samples The inventors' Dim1 assay was validated using clinical samples from infected and uninfected canine whole blood samples. Briefly, DNA was isolated from 300 μl of canine whole blood using the Qiagen DNeasy Blood and Tissue DNA column extraction kit according to the manufacturer's instructions. The DNA was eluted in 75 μl of ddH2O and 1 μl was used in each Dim1 real-time PCR assay. The data show no detection of D. immitis DNA in four uninfected dogs and three dogs infected with Brugia pahangi (Table 6). B. pahangi is a closely related filarial nematode parasite. Thus, no false positives or cross-reactivity with the feline / canine parasite B. pahangi were observed. Furthermore, no detection of D. immitis was seen in two dogs infected with 50 L3 D. immitis larvae 2.5 months prior to blood collection (Table 6). These two dogs had early prepatent infections without detectable microfilariae in the blood. The D. immitis parasite typically develops into immature adults and first invades the bloodstream approximately 70 days after infection [Kotani, T. & Powers, K. G. Am. J. Vet. Res. 43, 2199-2206 (1982)]. Thus, detection of D. immitis DNA in whole blood samples at 2.5 months post-infection was not necessarily expected as this was immediately after the first worms reached the bloodstream. High-level detection was seen in four dogs that showed patent infection with D. immitis (Table 6). The D. immitis Dim1 real-time PCR assay functioned as expected in clinical samples, showing no detection in unexposed and early prepatent dogs, no cross-reactivity in Brugia pahangi-infected dogs, and strong detection in dogs with patent infection and high microfilarial counts in blood samples.

[0161]

Table 6

[0162] (Example 6) Sensitivity of the Dim1 real-time PCR assay for the detection of prepatent infection using cell-free DNA The clinical sensitivity of our Dim1 real-time PCR assay for the detection of prepatent D. immitis infection was evaluated in two infected dogs (Dog #8 and Dog #9) using cell-free DNA (cfDNA) isolated from plasma. During the prepatent period, D. immitis worms start as L3 larvae (the infectious stage from mosquitoes) and then, approximately 70 days after infection, enter the host bloodstream as immature adults. When these immature adults enter the bloodstream, they can shed cells and DNA into the host bloodstream. Patency is reached approximately 6 - 7 months after infection, at which point these infections can be detected by current standard serological techniques that identify antigens shed by sexually mature adult females. During the prepatent period, adult female antigens are either absent or not present at a high enough concentration to be detected by current serological techniques. However, DNA shed from immature adults is present in the bloodstream as cell-free DNA (cfDNA) during this prepatent period. We demonstrated that D. immitis DNA can be detected in clinically prepatent infections using our D. immitis Dim1 real-time PCR assay as part of the total cfDNA fraction. Briefly, whole blood was collected from each dog every two weeks starting 3 months after infection with 50 D. immitis third-stage larvae (L3). Total cfDNA was isolated from dog plasma using the Thermofisher MagMAX™ Cell-Free DNA Isolation Kit according to the manufacturer's instructions. At each time point and for each dog, 2 μl of the isolated DNA was used as starting material and the elution volume was 30 μl. For our Dim1 real-time PCR assay, 5 μl of the eluate was used as template for each PCR reaction and each sample was tested in triplicate.

[0163] Data showed that D. immitis cfDNA was detected in plasma samples from all time points using the inventors' Dim1 real-time PCR assay in each of two artificially infected dogs, as indicated by mean Cq values of less than 40 (Table 7). Serological antigen detection of infection using the current standard point-of-care device (DiroCHEK® Antigen Test (Zoetis, Parsippany, NJ)) first occurred at 5 months and 5.5 months post-infection, respectively, for Dog #8 and Dog #9 in heat-treated serum (heat treatment of serum has been demonstrated to improve DiroCHEK assay sensitivity). In non-heat-treated serum, serological detection of antigen first occurred at 5 months and 7.5 months post-infection, respectively, for Dog #8 and Dog #9.

[0164] As shown in Table 7, D. immitis cfDNA was clearly detected in plasma prior to serological detection of antigen at 3, 3.5, 4, and 4.5 months post-infection, as indicated by Cq values of less than 40. These results demonstrate the ability of the inventors' D. immitis Dim1 real-time PCR assay to detect patent infection in dogs more than 2 months prior to antigen detection of infection. This provides an important implication for successful early drug treatment prior to patency.

[0165] [Table 7]

[0166] To evaluate whether the inventors' D. immitis Dim1 real-time PCR assay can produce false positive detection of cfDNA from canine plasma, two unexposed dogs and two B. pahangi-infected dogs were tested. As shown in Table 8, the data indicate that no signal was detected in any of the samples, indicating no false positive detection and no cross-reactivity / detection with the feline / canine parasite B. pahangi.

[0167]

Table 8

[0168] (Example 7) Use of magnetic nanoparticle technology and oligonucleotides to capture D. immitis Dim1 target DNA in buffer The inventors evaluated the ability of magnetic nanoparticle technology (magnetic beads) to efficiently capture D. immitis DNA in solution in conjunction with biotin-modified D. immitis-specific capture oligonucleotides. To mimic positive canine samples, 100 pg of D. immitis genomic DNA (gDNA) (Sample 1) as well as 100 pg each of D. immitis genomic gDNA and canine gDNA (Sample 2) were added to 200 μl of 1 M salt binding buffer. A set of 5'-biotinylated capture oligonucleotides was used to specifically target and hybridize to D. immitis gDNA in solution. Each pair of capture oligonucleotides was designed to be complementary to opposing DNA strands within the D. immitis Dim1 target DNA repeat, enabling capture of both strands of D. immitis DNA. Isolation of the captured DNA was achieved by ligating the Dim1 target DNA to the beads via biotin-streptavidin linkage to streptavidin-coated magnetic beads (Dynabeads™ M-270 Streptavidin, ThermoFisher) (Figure 2). After elution, the isolated DNA samples were enriched for D. immitis Dim1 target DNA. The recovered DNA was detected by the inventors' Dim1 repeat real-time PCR assay, and the results were compared to a standard curve (Figure 4) of known amounts of input D. immitis gDNA.

[0169] As shown in Table 9, the data show amplification and detection of recovered D. immitis gDNA in both test samples, as indicated by Cq values of 19.98 and 20.44, respectively. For each sample, the average Cq value is the average of 5 replicates per sample.

[0170] To determine the amount of DNA recovered from the solution, the inventors performed their D. immitis Dim1 real-time PCR assay using 10 picograms, 5 picograms, 2.5 picograms, and 1 picogram of input D. immitis gDNA and generated a standard curve using the average Cq values (Figure 4). Using the best-fit line formula, DNA recovery was determined to be 7.9 picograms per 5 μl of input template volume (Table 9, Sample 1) and 7.1 picograms per 5 μl of input template volume (Table 9, Sample 2). Considering a total DNA recovery volume of 50 μl for each sample, the total recovery is calculated to be 79 picograms and 71 picograms, respectively. These data show that this DNA capture method recovered 79% of D. immitis DNA from Table 9 Sample 1 (100 pg of added D. immitis DNA) and 71% of D. immitis DNA from Table 9 Sample 2 (100 pg of added D. immitis DNA and 100 pg of canine DNA). These results demonstrate the ability of this DNA capture method to efficiently recover specific DNA sequences from solution.

[0171] Furthermore, this specific capture method promotes enrichment of D. immitis target DNA, resulting in optimal sensitivity for detection using the inventors' Dim1 real-time PCR assay.

[0172]

Table 9

[0173] (Example 8) Use of Magnetic Nanoparticle Technology and Oligonucleotides to Capture D. immitis Dim1 Target DNA from Canine Plasma The inventors have shown that magnetic nanoparticle (magnetic bead) technology coupled with biotin-modified D. immitis-specific capture oligonucleotides can capture D. immitis The ability to efficiently capture DNA was further evaluated. To mimic positive canine plasma samples, 100 pg of D. immitis gDNA was added to 200 μl of plasma derived from unexposed dogs. 1× binding buffer supplemented with 100 pg each of D. immitis and canine DNA was used as a comparison for total DNA recovery. As shown in Table 10, the data show amplification and detection of recovered D. immitis DNA in both test samples, as indicated by Cq values of 19.79 (plasma sample) and 20.52 (1 M salt buffer), respectively. This data shows a Cq difference of less than 1, indicating similar recovery of input DNA in both samples. Therefore, this method is capable of efficiently capturing parasite DNA from plasma.

[0174]

Table 10

[0175] (Example 9) Sensitivity of the Dim1 real-time PCR assay for detecting patent infections using cell-free DNA The clinical sensitivity of the inventors' Dim1 real-time PCR assay embodiment for detecting patent microfilaremia-positive D. immitis infections was evaluated in three D. immitis-infected dogs (Dog #14, Dog #15, and Dog #16) using cell-free DNA ("cfDNA") isolated from canine plasma. Total cfDNA was isolated from canine plasma using the Thermofisher MagMAX™ Cell-Free DNA Isolation Kit (ThermoFisher, Waltham, Massachusetts, USA) according to the manufacturer's instructions. At each time point and for each dog, 2 ml of plasma was used as the starting material and the elution volume was 30 μl. 5 μl of the eluate was used as a template for each real-time PCR reaction using the primer and probe set p1Dim1, and each sample was tested in triplicate.

[0176] As shown in Table 11, D. immitis cfDNA was clearly detected in the plasma of patent microfilaria-positive dogs. These results demonstrate the presence of D. immitis cfDNA in plasma samples from dogs with patent infection and high microfilaria counts.

[0177]

Table 11

[0178] (Example 10) Strip test detection of Dim1 repetitive target DNA (SEQ ID NO: 2) The strip test detection sensitivity of D. immitis repetitive species-specific target DNA was evaluated using primer and probe set p4Dim1 (SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 30) (Figure 5) with serially diluted amounts (1 picogram, 100 femtograms, 10 femtograms, 1 femtogram, 100 attograms, 10 attograms, 1 attogram, 100 zeptograms) of D. immitis genomic DNA as a template. Briefly, PCR was performed using forward and reverse primers, SEQ ID NO: 9 and SEQ ID NO: 29, respectively, for 35 cycles. One DNA strand of the PCR product (the strand synthesized from the biotinylated PCR primer SEQ ID NO: 29) is biotinylated after PCR amplification. The PCR product is denatured (5 minutes at 95°C), and a 6-FAM labeled probe (SEQ ID NO: 30) is added to hybridize to the biotinylated DNA strand. Through lateral flow, the sample moves across the full length of the test strip. A visible positive result occurs when the biotinylated strand hybridized to the 6-FAM labeled probe binds to a band on the streptavidin-coated test strip (capture medium). A gold-labeled anti-fluorescein (6-FAM) antibody that binds to the 6-FAM labeled probe enables visualization of the Dim1 PCR product on the test strip.

[0179] The data indicate that the strip detection method consistently detected D. immitis genomic DNA down to 1 attogram of input D. immitis genomic DNA (Figure 6A). According to these data, the analytical sensitivity of the strip detection method is between 10 attograms and 1 attogram of input D. immitis genomic DNA, comparable to the embodiments of the Dim1 D. immitis real-time PCR assay (Table 1).

[0180] The inventors' Dim1 strip test detection assay was verified using clinical samples from infected and uninfected canine plasma samples. Briefly, total cfDNA was isolated from canine plasma using the Thermofisher MagMAX™ Cell-Free DNA Isolation Kit according to the manufacturer's instructions. For each canine sample, 2 ml of plasma was used as the starting material and the elution volume was 30 μl. 5 μl of the eluate was used as the template for each PCR reaction (35 cycles) using the primer and probe set p4Dim1.

[0181] Alternatively, parasite-specific cfDNA can be captured using a capture medium, such as a magnetic bead surface appropriately coated with a capture oligonucleotide that binds to the capture oligonucleotide. The oligonucleotide can first be bound to the appropriately treated capture medium and then hybridized to the target DNA, such as Dim1. The target DNA can be eluted from the beads using PCR amplification (35 cycles) with strip primers (SEQ ID NO: 9 and SEQ ID NO: 29). Here, the supernatant contains the PCR product containing one biotinylated strand. As shown above, a magnet can be used to remove the beads, the amplified target DNA can be recovered, and tested using a strip.

[0182] In either case, the PCR product can be denatured (5 minutes at 95°C), and a strip test probe, e.g., SEQ ID NO: 30, can be added to hybridize to the biotinylated DNA strand. As described above, via lateral flow, the sample moves across the full length of the test strip. When the biotinylated strand hybridized to the 6-FAM labeled probe binds to a band on the streptavidin-coated test strip (capture medium), a visible positive result occurs. The gold-labeled anti-fluorescein (6-FAM) antibody that binds to the 6-FAM labeled probe enables visualization of the Dim1 PCR product on the test strip.

[0183] The strip test detection assay functioned as expected in clinical samples, showed no detection in unexposed dogs, showed no cross-reactivity in Brugia pahangi-infected dogs, and showed strong detection in dogs with patent infection and high microfilaria counts in blood samples (Figs. 6B and 6C). See also Zaky et al. PLoS Negl Trop Dis. 2018 Nov 21;12(11).

[0184] References Akter, S., Nakao, R., Imasato, Y., Alam, M. Z. & Katakura, K. Potential of cell-free DNA as a screening marker for parasite infections in dog. Genomics 111, 906-912 (2019).

[0185] Albonico, F. et al. Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis. Vet. Parasitol. 200, 128-132 (2014).

[0186] Capelli, G. et al. Recent advances on Dirofilaria repens in dogs and humans in Europe. Parasites and Vectors 11, 1-21 (2018).

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[0209] Various embodiments of the present invention can be characterized by the potential claims (enumerated in the paragraphs following this paragraph and before the actual claims provided at the end of this application) that are listed below. These potential claims form part of the description of this application. Therefore, the subject matter of the following potential claims may be presented as the actual claims in subsequent proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Therefore, a decision not to present these potential claims in subsequent proceedings should not be construed as a dedication of that subject matter to the public.

[0210] Without limitation, potential subject matter that may be claimed (preceded by the letter "P" to avoid confusion with the actual claims presented below) includes the following: Claim P1. A device for diagnosing parasitic infections in an animal host, comprising a PCR measurement device including a thermocycler, wherein the PCR measurement device is configured to target repetitive sequences in the DNA of the parasite in a tissue sample obtained from the infected animal host. Claim P2. The device according to claim P1, wherein the parasite is selected from the group consisting of Dirofilaria immitis and Dirofilaria repens. Claim P3. The device according to any one of claims P1 to P2, wherein the repetitive sequence is selected to provide sensitivity and selectivity for the parasite. Claim P4. The device according to any one of claims P1 to P3, wherein the PCR measurement device includes an optical reading mechanism. Claim P5. The device according to claim P4, wherein the accompanying optical reading mechanism includes a test strip having a visible band. Claim P6. The device according to any one of claims P1 to P5, wherein the tissue sample is selected from the group consisting of plasma, serum, and whole blood obtained from the infected animal host. Claim P7. The device according to any one of claims P1 to P6, wherein the tissue sample is derived from an infected mosquito. Claim P8. A method for treating a mammalian host suspected of being infected with a parasite, comprising: (a) providing a sample from the mammalian host suspected of being infected with the parasite; and (b) causing detection of the presence of the parasite in the sample, the detection comprising: (i) making DNA available from the sample; (ii) mixing the sample with a labeled DNA complementary to the target DNA; and (iii) detecting the target DNA using PCR including the steps including, wherein if the presence of the parasite is detected as a result of the steps causing detection, the mammalian host is administered a treatment effective to reduce or eliminate the parasite infection. Claim P9. The labeled DNA is a. identifying the target DNA, wherein the target DNA is repetitive DNA uniquely possessed by the parasite, and the identifying step comprises: i. obtaining genomic DNA sequence reads from the parasite, ii. trimming each sequence read to generate a set of trimmed sequence reads of equal length, iii. comparing each sequence in the set of reads with all other sequence reads in the set of reads to form read pairs, wherein each read of a given pair shares at least 90% similarity with the other read of the pair over at least 55% of its length. iv. evaluating the lead pairs to identify a candidate set of sequences in the genome of the parasite that are putatively present at high copy numbers in the genome; and v. selecting, from the candidate set of sequences, a final sequence uniquely held by the parasite Steps; and b. using the final sequence uniquely held by the parasite to cause synthesis of the labeled DNA The method according to claim P8, which is produced using a process comprising, wherein the labeled DNA is complementary to the identified repetitive DNA uniquely held by the parasite. Claim P10. The method according to any one of claims P8 to P9, wherein the parasite is selected from the group consisting of D. immitis and D. repens. Claim P11. The method according to claim P10, wherein the parasitic infection is patent. Claim P12. The method according to claim P10, wherein the parasitic infection is pre-patent. Claim P13. The method according to any one of claims P8 to P12, wherein the tissue sample is selected from the group consisting of plasma, serum, and whole blood obtained from an infected mammalian host. Claim P14. The method according to any one of claims P8 to P13, wherein the mammalian host is a dog. Claim P15. The method according to any one of claims P8 to P14, wherein the labeled DNA comprises a conjugation moiety. Claim P16. The method according to claim P15, wherein the conjugation moiety is biotin. Claim P17. The method according to any one of claims P8 to P16, wherein the target DNA is isolated from the mixture using a capture medium that binds to the capture DNA. Claim P18. The method according to claim P17, wherein the capture medium is selected from the group consisting of magnetic beads and test strips. Claim P19. The method according to any one of claims P17 to P18, wherein the capture medium is coated with streptavidin. Claim P20. The method according to any one of claims P8 to P19, wherein the PCR is real-time PCR using a primer and a probe set. Claim P21. The method according to claim P20, wherein the parasite is D.immitis, and the primer and probe set is selected from the group consisting of p1Dim1, p2Dim1, and p3Dim1. Claim P22. The method according to claim P20, wherein the parasite is D.repens, and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1, and p3Dre1. Claim P23. The method according to any one of claims P8 to P21, wherein the parasite is D.immitis and the target DNA is Dim1. Claim P24. The method according to any one of claims P8 to P21 and P23, wherein the parasite is D.immitis and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4. Claim P25. The method according to any one of claims P8 to P20 and P22, wherein the parasite is D.repens and the target DNA is Dre1. Claim P26. The method according to any one of claims P8 to P20, P22, and P25, wherein the parasite is D.repens and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8. Claim P27. The method according to any one of claims P8 to P26, wherein the treatment is selected from the group consisting of melarsomine, ivermectin, doxycycline, moxidectin, and combinations thereof. Claim P28. A kit for detecting parasite infection in an animal host, comprising: a) labeled DNA complementary to repetitive species-specific parasite target DNA b) a capture medium that binds to the DNA, and c) a set of written instructions for detecting the parasitic infection A kit comprising. Claim P29. The kit according to claim P28, wherein the parasite is selected from the group consisting of D. immitis and D. repens. Claim P30. The kit according to any one of claims 28 to 29, wherein the labeled DNA comprises a conjugation moiety. Claim P31. The kit according to claim P30, wherein the conjugation moiety is biotin. Claim P32. The kit according to any one of claims P28 to P31, wherein the capture medium is selected from the group consisting of magnetic beads, test strips, and combinations thereof. Claim P33. The kit according to any one of claims P28 to P32, wherein the capture medium is coated with streptavidin. Claim P34. The kit according to any one of claims P28 to P33, comprising a primer and a probe set. Claim P35. The kit according to any one of claims P28 to P34, wherein the parasite is D. immitis and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4. Claim P36. The kit according to any one of claims P28 to P35, wherein the parasite is D. immitis and the target DNA is Dim1. Claim P37. The kit according to claim P34, wherein the parasite is D. immitis and the primer and probe set are selected from the group consisting of p1Dim1, p2Dim1, p3Dim1, and p4Dim1. Claim P38. The kit according to any one of claims 28 to 34, wherein the parasite is D. repens and the labeled DNA is a set of capture oligonucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8. Claim P39. The kit according to any one of claims P28 to P34 and P38, wherein the parasite is D. repens and the target DNA is Dre1. Claim P40. The kit according to claim P34, wherein the parasite is D. repens and the primer and probe set is selected from the group consisting of p1Dre1, p2Dre1 and p3Dre1.

[0211] The above embodiments of the present invention are intended to be illustrative only; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the invention as defined in any appended claims.

Claims

1. A kit for detecting Dirofilaria immitis infection in an animal host, comprising: a) a probe complementary to a repetitive species-specific Dirofilaria immitis sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, said probe comprising one or more moieties for facilitating its detection; and b) a capture medium configured to bind to the capture DNA The kit.

2. The kit according to claim 1, comprising a first capture oligonucleotide complementary to SEQ ID NO: 1 and a second capture oligonucleotide complementary to SEQ ID NO: 2, each of said first capture oligonucleotide and said second capture oligonucleotide comprising a conjugation moiety.

3. The kit according to claim 2, wherein the conjugation moiety is biotin.

4. The kit according to any one of claims 1 to 3, wherein the capture medium is selected from the group consisting of magnetic beads, test strips, and combinations thereof.

5. The kit according to any one of claims 1 to 4, wherein the capture medium is coated with streptavidin.

6. The kit according to any one of claims 1 to 5, comprising a set of primers.

7. The kit according to claim 2, or any one of claims 3 to 6 when dependent directly or indirectly on claim 2, wherein the first capture oligonucleotide is SEQ ID NO: 3 and the second capture oligonucleotide is SEQ ID NO:

4.

8. The kit according to any one of claims 1 to 7, wherein the probe is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:

30.

9. The set of primers of the kit is SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 9 and SEQ ID NO: 21; SEQ ID NO: 9 and SEQ ID NO: 23; and SEQ ID NO: 9 and SEQ ID NO: 29 The kit according to claim 6, or any one of claims 7 to 8 when dependent directly or indirectly on claim 6, selected from the group consisting of.

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