Indian Patents. 257150:“A PRIMER SET AND A PROCESS FOR THE DETECTION OF L3 LARVAL STAGE OF WUCHERERIA BANCROFTI"

Title of Invention	“A PRIMER SET AND A PROCESS FOR THE DETECTION OF L3 LARVAL STAGE OF WUCHERERIA BANCROFTI"
Abstract	The present invention relates to a process for the diagnosis of infective stage (L3) larva of filarial parasite Wuchereria bancrofti, in mosquito vector, Culex quinquefasciatus. The present invention also relates to a set of following novel primers: Forward primer 5" -GAG TCG TTT GOT TGG GGA TA- 3". Reverse primer 5"- TCT TCT TGC CCA GTA CAG CA- 3".

Full Text	FIELD OF THE INVENTION; The present invention relates to a process for the diagnosis of infective stage (L3) larva of filarial parasite Wuchereria bancrofti, in mosquito vector, Culex quinquefasciatus. BACKGROUND AND PRIOR ART; The infective (L3) stage of Wuchereria bancrofti is the one that initiates infection in human after getting itself transmitted through the bite of mosquitoes. Detection/diagnosis of the presence of L3 stage of the filarial parasite is important for monitoring the transmission of infection in a filariasis endemic area, delimiting filariasis endemic areas and monitoring control operations such as recently launched Global Programme for Elimination of Lymphatic Filariasis. Currently, it is being done by conventional technique of dissection of vector mosquitoes followed by examination under microscope (Sasa, 1976). Alive mosquito brought from field for examination of infection is knocked down using ether and then dissected in saline solution with the help of needles and forceps. The skilled technician will then observe under microscope for the presence of parasite stages and records the observations. Hence, this technique is cumbersome, needs experienced person to observe the infection, subjective and not amenable for large scale screening of vectors. Therefore, there is a need to develop alternative diagnostic techniques. Our invention relates to a process which is not cumbersome, does not require live mosquito samples, and is amenable for large scale screening of mosquito vectors unlike the conventional technique. Recently, species specific molecular biological methods such as polymerase chain reaction (PCR) assays have been developed for detecting infection with (all stages: mf, LI, L2, L3) lymphatic filarial parasites viz., Wuchereria bancrofti and Brugia malayi in Cx. quinquefasciatus and Mansonioides mosquitoes, respectively (Zhong et a/., 1996; Lizotte et al, 1994). Although these assays can distinguish between the two parasites of lymphatic filariasis, they do not differentiate between different developmental stages of the two parasites. OBJECTS OF THE INVENTION; An object of the invention is to propose a process for the diagnosis of infective stage (L3) larva of filarial parasite, Wuchereria bancrofti in vector mosquito, Culex quinque-fasciatus. Another object of the invention is to propose a process for the diagnosis of infective stage (L3) larva of filarial parasites in individual vector mosquito with 90-100% sensitivity and specificity. Yet another object of the invention is to propose a process for the diagnosis of infective stage (L3) larva of filarial parasites in vector mosquitoes in pools with 90-100% sensitivity and specificity. SUMMARY OF THE INVENTION; The present invention relates to a process for the diagnosis or detection of infective stage (L3) larva of filarial parasite, Wuchereria bancrofti in vector mosquito Culex quinquefasciatus. Infected mosquito is homogenized with denaturing agent, from which RNA is extracted using organic solvents. First strand cDNA is synthesized by reverse transcription reaction from extracted RNA, by using oligo-dT primer and reverse transcriptase enzyme. Stage-specific primers (forward and reverse) are used to amplify the first strand cDNA to second strand cDNA, which is run on agarose gel, stained with ethidium bromide, observed for a for a band of 112 bp on a UV transilluminator. BRIEF DESCRIPTION OF THE DRAWINGS: FIG. 1. Process of extracting RNA using TRI-REAGENT FIG. 2. Process of synthesizing a) first strand cDNA and b) second strand cDNA using RNA. FIG. 3. Sensitivity of the assay in the detection of infective stage parasite in mosquito. DETAILED DESCRIPTION OF THE INVENTION; Accordingly the present invention provides a method of diagnosis or detection of infective stage (L3) larva of filarial parasite, Wuchereria bancrofti in vector mosquito Culex quinquefasciatus. An embodiment of the invention is a process for the detection of infective (L3) stage larva of filarial parasite, Wuchereria bancrofti in vector mosquitoes comprising subjecting the mosquito samples to TRI REAGENT for homogenization, followed by RNA extraction, to obtain RNA pellet solubilized in DEPC treated water for synthesizing first strand cDNA by reverse transcription reaction, utilizing the product of the said reverse transcription for the synthesis of second strand cDNA with stage specific forward and reverse primers by polymerase chain reaction, running the product of the said second strand synthesis electrophoretically on agarose gel and staining with ethidium bromide to obtain a band of 112 bp. Another embodiment the mosquitoes are suspended in 1 ml of TRI REAGENT. In another embodiment of the invention mosquitoes with infective (L3) stage parasites are ground and homogenized in TRIREAGENT using polypropylene micropestles. In yet another embodiment of the invention the mosquito homogenate is added with 200 ul of chloroform, shaken vigorously, allowed to stand at room temperature (25-30°C) for 15 min and centrifuged at 12,000 rpm for 15 minutes at 4°C and the aqueous phase is transferred to a fresh tube. In yet another embodiment of the invention the aqueous phase is added with 500 ul of isopropanol, mixed, allowed to stand at room temperature (25-30°C) for 10 min, centrifuged at 12,000 rpm at 4°C for 10 minutes and the supernatant is removed and discarded. In another embodiment of the invention the pellet containing RNA is suspended in 1 ml of 75% ethanol, centrifuged at 12,000 rpm for 5 minutes, pellet retained, supernatant removed and discarded. In still another embodiment of the invention the RNA pellet is resuspended in RNase free water. Another embodiment of the invention is synthesis of first strand cDNA and second strand cDNA in a two stage process. In an embodiment of the invention first stage comprises synthesis of first strand cDNA, by transferring RNA (3-7µl) to reverse transcription reaction mixture containing oligodT primer (l0µM), reverse transcriptase enzyme (2-6 units), RT buffer (Ix), dNTPs (0.2-0.7 mM each dNTP), RNase inhibitor (5-15 units) and RNase free water in total volume of 30 µl at 30-40°C for 1-2 hours. In another embodiment of the invention second stage comprises synthesis of second strand cDNA, by transferring 2-6µl of the product obtained from the first stage to polymerase chain reaction mixture containing forward and reverse primers (5-15 pM each), Tag DNA polymerase (1-4 units), buffer (Ix), dNTPs (5-15 mM each dNTP), sterile double distilled water to make up a final volume of 20-50µl. In another embodiment of the invention polymerase chain reaction is carried out in a thermal cycler with temperature cycles of 94°C for 5-10 min with 30-35 cycles of 94°C for 1-2 min, 55°C for 1-2 min and 72°C for 1-2 min and a final extension of 72 °C for 7-10 min. In another embodiment of the invention running the product of the said second strand synthesis is carried out on agarose gel electrophoretically and stained with ethidium bromide to obtain a band of 112 bp. In another embodiment of the invention the said gel electrophoresis is carried out at a concentration of 1-2% Agarose in TAE buffer. In still another embodiment of the invention the said ethidium bromide staining of the agarose gel is carried out at a concentration of 0.5µg/ml in water. An embodiment of the invention is a forward primer 5' -GAG TCG TTT GGT TGGGGATA-3'. Another embodiment of the invention is a reverse primer 5'- TCT TCT TGC CCA GTACAGCA-3'. An embodiment of the invention is that the detection of infective stage requires comparatively lesser skill. Yet another embodiment of the invention is that the species and stage of the parasite is decided by the technique itself and hence objective. In another embodiment of the invention a single parasite present in the mosquito is detected even in the presence of large amount of mosquito tissues. In yet another embodiment of the invention mass screening of pools of mosquitoes by RT-PCR assay is carried out. Adopting robotized system is also possible. The present invention employs a process in which a novel Wuchereria bancrofti L3-specific probe is employed in reverse transcription PCR for the detection/diagnosis of the presence of infective (L3) stage in Cx. quinquefasciatus. According to this invention there is provided a process for the detection of infective stage (L3) larva of filarial parasites in vector mosquitoes herein described which comprises in subjecting the mosquito samples to TRI REAGENT (phenol+guanidine thiocyanate) for homoge-nization, followed by RNA extraction by phase separation with chloroform and then precipitation using isopropanol and washing the extracted RNA with 75% ethanol and after drying the RNA pellet, solubilizing in DEPC treated water. The process details are discussed in FIG. 1 and FIG. 2 wherein following RNA extraction, first strand cDNA is synthesized from the extracted RNA (1.0 - 2.0 ug) using oligo-dT primer (0.5-l.0µM), Ix buffer RT, Reverse Transcriptase enzyme (2-4 units), dNTPs (0.1-0.5 mM each dNTP), RNase inhibitor (5-10 units) and RNase free water in total volume of 20-25 µ1 at 40 to 50°C for 1-2 hours. The second strand cDNA is synthesized using 3-µl of single strand cDNA along with stage specific primer (10-15 pM each) forward 5' -GAG TCG TTT GGT TGG GGA TA- 3' and reverse 5'- TCT TCT TGC CCA GTA CAG CA- 3'), Taq DNA polymerase (4 units), dNTPs (10-15mM each dNTP) sterile double distilled water to make up a final volume of 30µl, with temperature cycles of 94 °C for 5-10 min with 30-35 cycles of 94°C for 1-2 min, 55-°C for 1-2 min and 72°C for 1-2 min and a final extension of 72 °C for 10-15 min. The products are run on agarose gel (1-2% in TAB buffer), stained with Ethidium bromide (0.1-0.5µg/ml in water) observed for a band of 112 bp. EXAMPLES: Samples of mosquitoes with filarial parasite at the stage of mf or LI or L2 or L3 and without any parasite are coded and processed further as described herein above for determining the stage specificity of the novel primer employed. The process is specific in detecting infective stage (L3) parasite by giving positive signal with a band of 112 bp and does not give any amplification when other stages are present and the specificity accounts to 98% (Table 1). Samples of individual mosquito with infective (L3) stage parasite and without any parasites are blinded and sensitivity is assessed by processing the samples as described herein above. All the mosquito samples having infective (L3) stage parasite are positive by indicating the presence of all2 bp band (Table 2). All uninfected mosquitoes showed negative signal indicating the absence of the above said band. In order to make the process cost effective, the mosquito samples are prepared by pooling ten mosquitoes with infective stage parasite and subjected to the process described herein above (Table 3). Table 1 (Table Removrd) Specificity of the assay by the process of present invention Table 2. (Table Removrd) Sensitivity of the assay by the process of present invention (Table Removrd) Table 3. (Table Removrd) Sensitivity of the assay in pooled samples by the process of present invention

Full Text

FIELD OF THE INVENTION;
The present invention relates to a process for the diagnosis of infective stage (L3) larva of filarial parasite Wuchereria bancrofti, in mosquito vector, Culex quinquefasciatus.
BACKGROUND AND PRIOR ART;
The infective (L3) stage of Wuchereria bancrofti is the one that initiates infection in human after getting itself transmitted through the bite of mosquitoes. Detection/diagnosis of the presence of L3 stage of the filarial parasite is important for monitoring the transmission of infection in a filariasis endemic area, delimiting filariasis endemic areas and monitoring control operations such as recently launched Global Programme for Elimination of Lymphatic Filariasis. Currently, it is being done by conventional technique of dissection of vector mosquitoes followed by examination under microscope (Sasa, 1976). Alive mosquito brought from field for examination of infection is knocked down using ether and then dissected in saline solution with the help of needles and forceps. The skilled technician will then observe under microscope for the presence of parasite stages and records the observations. Hence, this technique is cumbersome, needs experienced person to observe the infection, subjective and not amenable for large scale screening of vectors. Therefore, there is a need to develop alternative diagnostic techniques. Our invention relates to a process which is not cumbersome, does not require live mosquito samples, and is amenable for large scale screening of mosquito vectors unlike the conventional technique. Recently, species specific molecular biological methods such as polymerase chain reaction (PCR) assays have been developed for detecting infection with (all stages: mf, LI, L2, L3) lymphatic filarial parasites viz., Wuchereria bancrofti and Brugia malayi in Cx. quinquefasciatus and Mansonioides mosquitoes, respectively (Zhong et a/., 1996; Lizotte et al, 1994). Although these assays can distinguish between the two parasites of lymphatic filariasis, they do not differentiate between different developmental stages of the two parasites.
OBJECTS OF THE INVENTION;
An object of the invention is to propose a process for the diagnosis of infective stage (L3) larva of filarial parasite, Wuchereria bancrofti in vector mosquito, Culex quinque-fasciatus.
Another object of the invention is to propose a process for the diagnosis of infective stage (L3) larva of filarial parasites in individual vector mosquito with 90-100% sensitivity and specificity.
Yet another object of the invention is to propose a process for the diagnosis of infective stage (L3) larva of filarial parasites in vector mosquitoes in pools with 90-100% sensitivity and specificity.
SUMMARY OF THE INVENTION;
The present invention relates to a process for the diagnosis or detection of infective stage (L3) larva of filarial parasite, Wuchereria bancrofti in vector mosquito Culex quinquefasciatus. Infected mosquito is homogenized with denaturing agent, from which RNA is extracted using organic solvents. First strand cDNA is synthesized by reverse transcription reaction from extracted RNA, by using oligo-dT primer and reverse transcriptase enzyme. Stage-specific primers (forward and reverse) are used to amplify the first strand cDNA to second strand cDNA, which is run on agarose gel, stained with ethidium bromide, observed for a for a band of 112 bp on a UV transilluminator.
BRIEF DESCRIPTION OF THE DRAWINGS:
FIG. 1. Process of extracting RNA using TRI-REAGENT
FIG. 2. Process of synthesizing a) first strand cDNA and b) second strand cDNA using RNA.
FIG. 3. Sensitivity of the assay in the detection of infective stage parasite in mosquito.
DETAILED DESCRIPTION OF THE INVENTION;
Accordingly the present invention provides a method of diagnosis or detection of infective stage (L3) larva of filarial parasite, Wuchereria bancrofti in vector mosquito Culex quinquefasciatus.
An embodiment of the invention is a process for the detection of infective (L3) stage larva of filarial parasite, Wuchereria bancrofti in vector mosquitoes comprising subjecting the mosquito samples to TRI REAGENT for homogenization, followed by RNA extraction, to obtain RNA pellet solubilized in DEPC treated water for synthesizing first strand cDNA by reverse transcription reaction, utilizing the product of the said reverse transcription for the synthesis of second strand cDNA with stage specific forward and reverse primers by polymerase chain reaction, running the product of the said second strand synthesis electrophoretically on agarose gel and staining with ethidium bromide to obtain a band of 112 bp.
Another embodiment the mosquitoes are suspended in 1 ml of TRI REAGENT.
In another embodiment of the invention mosquitoes with infective (L3) stage parasites are ground and homogenized in TRIREAGENT using polypropylene micropestles.
In yet another embodiment of the invention the mosquito homogenate is added with 200 ul of chloroform, shaken vigorously, allowed to stand at room temperature (25-30°C) for 15 min and centrifuged at 12,000 rpm for 15 minutes at 4°C and the aqueous phase is transferred to a fresh tube.
In yet another embodiment of the invention the aqueous phase is added with 500 ul of isopropanol, mixed, allowed to stand at room temperature (25-30°C) for 10 min, centrifuged at 12,000 rpm at 4°C for 10 minutes and the supernatant is removed and discarded.
In another embodiment of the invention the pellet containing RNA is suspended in 1 ml of 75% ethanol, centrifuged at 12,000 rpm for 5 minutes, pellet retained, supernatant removed and discarded.
In still another embodiment of the invention the RNA pellet is resuspended in RNase free water.
Another embodiment of the invention is synthesis of first strand cDNA and second strand cDNA in a two stage process.
In an embodiment of the invention first stage comprises synthesis of first strand cDNA, by transferring RNA (3-7µl) to reverse transcription reaction mixture containing oligodT primer (l0µM), reverse transcriptase enzyme (2-6 units), RT buffer (Ix), dNTPs (0.2-0.7 mM each dNTP), RNase inhibitor (5-15 units) and RNase free water in total volume of 30 µl at 30-40°C for 1-2 hours.
In another embodiment of the invention second stage comprises synthesis of second strand cDNA, by transferring 2-6µl of the product obtained from the first stage to polymerase chain reaction mixture containing forward and reverse primers (5-15 pM each), Tag DNA polymerase (1-4 units), buffer (Ix), dNTPs (5-15 mM each dNTP), sterile double distilled water to make up a final volume of 20-50µl.
In another embodiment of the invention polymerase chain reaction is carried out in a thermal cycler with temperature cycles of 94°C for 5-10 min with 30-35 cycles of 94°C for 1-2 min, 55°C for 1-2 min and 72°C for 1-2 min and a final extension of 72 °C for 7-10 min.
In another embodiment of the invention running the product of the said second strand synthesis is carried out on agarose gel electrophoretically and stained with ethidium bromide to obtain a band of 112 bp.
In another embodiment of the invention the said gel electrophoresis is carried out at a concentration of 1-2% Agarose in TAE buffer.
In still another embodiment of the invention the said ethidium bromide staining of the agarose gel is carried out at a concentration of 0.5µg/ml in water.
An embodiment of the invention is a forward primer 5' -GAG TCG TTT GGT TGGGGATA-3'.
Another embodiment of the invention is a reverse primer 5'- TCT TCT TGC CCA GTACAGCA-3'.
An embodiment of the invention is that the detection of infective stage requires comparatively lesser skill.
Yet another embodiment of the invention is that the species and stage of the parasite is decided by the technique itself and hence objective.
In another embodiment of the invention a single parasite present in the mosquito is detected even in the presence of large amount of mosquito tissues.
In yet another embodiment of the invention mass screening of pools of mosquitoes by RT-PCR assay is carried out. Adopting robotized system is also possible.
The present invention employs a process in which a novel Wuchereria bancrofti L3-specific probe is employed in reverse transcription PCR for the detection/diagnosis of the presence of infective (L3) stage in Cx. quinquefasciatus. According to this invention there is provided a process for the detection of infective stage (L3) larva of filarial parasites in vector mosquitoes herein described which comprises in subjecting the mosquito samples to TRI REAGENT (phenol+guanidine thiocyanate) for homoge-nization, followed by RNA extraction by phase separation with chloroform and then precipitation using isopropanol and washing the extracted RNA with 75% ethanol and after drying the RNA pellet, solubilizing in DEPC treated water.
The process details are discussed in FIG. 1 and FIG. 2 wherein following RNA extraction, first strand cDNA is synthesized from the extracted RNA (1.0 - 2.0 ug) using oligo-dT primer (0.5-l.0µM), Ix buffer RT, Reverse Transcriptase enzyme (2-4 units), dNTPs (0.1-0.5 mM each dNTP), RNase inhibitor (5-10 units) and RNase free water in total volume of 20-25 µ1 at 40 to 50°C for 1-2 hours. The second strand cDNA is synthesized using 3-µl of single strand cDNA along with stage specific primer (10-15 pM each) forward 5' -GAG TCG TTT GGT TGG GGA TA- 3' and reverse 5'- TCT TCT TGC CCA GTA CAG CA- 3'), Taq DNA polymerase (4 units), dNTPs (10-15mM each dNTP) sterile double distilled water to make up a final volume of 30µl, with temperature cycles of 94 °C for 5-10 min
with 30-35 cycles of 94°C for 1-2 min, 55-°C for 1-2 min and 72°C for 1-2 min and a final extension of 72 °C for 10-15 min. The products are run on agarose gel (1-2% in TAB buffer), stained with Ethidium bromide (0.1-0.5µg/ml in water) observed for a band of 112 bp.
EXAMPLES:
Samples of mosquitoes with filarial parasite at the stage of mf or LI or L2 or L3 and without any parasite are coded and processed further as described herein above for determining the stage specificity of the novel primer employed. The process is specific in detecting infective stage (L3) parasite by giving positive signal with a band of 112 bp and does not give any amplification when other stages are present and the specificity accounts to 98% (Table 1).
Samples of individual mosquito with infective (L3) stage parasite and without any parasites are blinded and sensitivity is assessed by processing the samples as described herein above. All the mosquito samples having infective (L3) stage parasite are positive by indicating the presence of all2 bp band (Table 2). All uninfected mosquitoes showed negative signal indicating the absence of the above said band.
In order to make the process cost effective, the mosquito samples are prepared by pooling ten mosquitoes with infective stage parasite and subjected to the process described herein above (Table 3).
Table 1
(Table Removrd)
Specificity of the assay by the process of present invention
Table 2.
(Table Removrd)
Sensitivity of the assay by the process of present invention
(Table Removrd)
Table 3.
(Table Removrd)
Sensitivity of the assay in pooled samples by the process of present
invention

Documents:

2793-del-2006-Abstract-(17-09-2012).pdf

2793-del-2006-Abstract-(31-08-2012).pdf

2793-del-2006-abstract.pdf

2793-del-2006-Claims-(17-09-2012).pdf

2793-del-2006-Claims-(31-08-2012).pdf

2793-del-2006-Correspondence Others-(11-04-2012).pdf

2793-DEL-2006-Correspondence Others-(12-03-2012).pdf

2793-DEL-2006-Correspondence Others-(22-03-2011).pdf

2793-del-2006-Correspondence Others-(31-08-2012).pdf

2793-del-2006-Correspondence-others-(15-01-2013).pdf

2793-DEL-2006-Correspondence-Others-(17-03-2011).pdf

2793-del-2006-Correspondence-Others-(17-09-2012).pdf

2793-del-2006-correspondence-others-1.pdf

2793-DEL-2006-Correspondence-Others.pdf

2793-DEL-2006-Description (Complete).pdf

2793-del-2006-description (provisional).pdf

2793-DEL-2006-Drawings-(17-03-2011).pdf

2793-del-2006-drawings.pdf

2793-del-2006-Form-1-(17-09-2012).pdf

2793-del-2006-Form-1-(31-08-2012).pdf

2793-DEL-2006-Form-1.pdf

2793-del-2006-form-18.pdf

2793-del-2006-Form-2-(17-09-2012).pdf

2793-del-2006-Form-2-(31-08-2012).pdf

2793-del-2006-form-2.pdf

2793-del-2006-form-26.pdf

2793-del-2006-form-3.pdf

2793-del-2006-form-5.pdf

2793-del-2006-GPA-(11-04-2012).pdf

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Patent Number

257150

Indian Patent Application Number

2793/DEL/2006

PG Journal Number

37/2013

Publication Date

13-Sep-2013

Grant Date

06-Sep-2013

Date of Filing

26-Dec-2006

Name of Patentee

INDIAN COUNCIL OF MEDICAL RESEARCH

Applicant Address

V. RAMALINGASWAMI BHAWAN, ANSARI NAGAR, POST BOX 4911, NEW DELHI-110029, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	HOTI, S.L	VECTOR CONTROL RESEARCH CENTRE, INDIRA NAGAR, PONDICHERRY-605006,INDIA
2	VASUKI, V	VECTOR CONTROL RESEARCH CENTRE, INDIRA NAGAR, PONDICHERRY-605006,INDIA

PCT International Classification Number

G01N33/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA