WO2014003583A2

WO2014003583A2 - Detection of pathogens

Info

Publication number: WO2014003583A2
Application number: PCT/PH2013/000016
Authority: WO
Inventors: Raul V. DESTURA; Joy Ann G. PETRONIO; Carmencita C. PADILLA; Ricky B. VINARAO; Kristine Marie G. FLORES; Jesus Emmanuel A.D. SAVILLEJA; Sharie Keanne C. GANCHUA
Original assignee: University Of The Philippines Diliman
Priority date: 2012-06-26
Filing date: 2013-06-26
Publication date: 2014-01-03
Also published as: WO2014003583A3; WO2014003583A8; WO2014003583A4

Abstract

The present invention relates to a methods and diagnostic kits for the rapid detection of different pathogens employing the loop-mediated isothermal amplification platform (LAMP) that may be performed or used even outside the laboratory. The said invention uses novel primer combinations for' sequences within the housekeeping genes of the target organism.

Description

DETECTION OF PATHOGENS

Technical Field This invention relates to the field of detection of pathogens in samples using loop-mediated isothermal amplification (LAMP) assay.

Background of the Invention According to the "First WHO report on neglected tropical diseases", neglected tropical diseases affect the lives of a billion people worldwide and threaten the health of millions more. Together with poverty, they weaken impoverished populations and impede global public health outcomes (cf. http://www.academicjournals.org/ajfs/pdf/Pdf2008/Jul/Deguo%20et%20al.pdf). Among the diseases that need to be diagnosed, monitored and controlled are dengue, tuberculosis, typhoid fever, leptospirosis and schistosomiasis.

Dengue fever and dengue haemorrhagic fever (DF/DHF) has emerged as the most important arboviral disease of mankind in terms of both morbidity and mortality. Although the majority of infections with dengue viruses (DENV) are asymptomatic, the wide spectrum of disease ranges from mild, self-limited dengue fever to severe and potentially life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Chakravarti, et al. 2006; Domingo, et al., 2006; Yong, et al., 2007). The dengue virus belongs to the family of Flaviviridae, and it consists of four closely- related but antigenically distinct serotypes (DENV- 1 , DENV-2, DENV-3, and DENV-4) . Mycobacteria are aerobic, non-spore forming, non-motile, single-cell bacteria. Of more than

40 currently recognized species of mycobacteria, Mycobacterium tuberculosis (MTB) is the leading cause of death world-wide that can be attributed to a single infectious disease agent. Tubeculosis (TB) is the disease caused by the bacteria of the MTB complex: M. tuberculosis, M. bovis and M. africanum. The World Health Organization (WHO) cites TB as the single most important fatal infection with 9.4 million new cases and 1.3 million deaths per year, 95% of which are in developing countries (WHO, 2009). The Philippines ranks 9^th in terms of TB burden (DOH, 2010) with 480,000 prevalence and 280,000 incidence of TB in the year 2009 (WHO, 2010). According to the Philippine Department of Health (DOH) of the Philippines (2005), TB continues to be the 6^th leading cause of morbidity and mortality among Filipinos.

Since the 1980s, many other mycobacterial, known as non-tuberculous mycobacterial (NTM) or mycobacteria other than tuberculosis (MOTT), have also been associated with human disease with increasing frequency worldwide. Non-tuberculous mycobacteria have been reported to cause localized or disseminated disease depending on local predisposition and/or degree of immuiie deficit. In non- HIV patients, different NTM may cause localized pulmonary disease, adenitis, soft tissue infections, infections of joints/bones, bursae, skin ulcers and generalized disease in individuals like leukaemia, transplant patients etc. In AIDS patients the manifestations may range from localized to disseminated disease. Clinical features will include local organ specific signs and symptoms to persistent high grade fever, night sweats, anaemia and weight loss in addition to nonspecific symptoms of malaise, anorexia, diarrhoea, myalgia and occasional painful adenopathy (Katoch, 2004). Discrimination between MTB and MOTT is very important as the treatment strategy for tuberculosis and other mycobacterioses is very different.

On the other hand, the salmonellae are members of the family enterobacteriaceae which is under the gamma proteobacteria. Species belonging to this group is responsible for enteritis and numerous worldwide foodborne outbreaks (Trafny et al., 2006). Currently, the Salmonella are divided into two species Salmonella enterica and Salmonella bongori (McQuiston et al., 2008). Salmonella enterica is further divided into six subspecies by multilocus enzyme electrophoresis (MLEE) (Boyd et al., 1996). Further classification of the subspecies was based on the O antigen of the lipopolysaccharide and the H antigen of the flagella. The frequently encountered species of salmonella are Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi both of which can cause typhoid fever (Ochiai et al., 2005).

Yet another disease, typhoid fever, has been reported by the WHO to have an estimated global incidence of typhoid fever reached 21 million (Wain and Hosoglu, 2008) and 7 million of which occurs in Southeast Asia with 200,000 deaths (Bhutta, 2006). In the Philippines the fatality incidence obtained in the year 2005 was 1.8 per 100,000 people (Kawano et al., 2007).

Leptospirosis is a global zoonosis that is widespread in humid tropical and subtropical regions and is considered as a re-emerging infectious disease. It is known to be well-represented zoonotic infection which occurs in humans as well as in animals. Infected animals excrete the organism in their urine and contaminate the environment. The common mode of transmission of the infection in humans include direct contact with contaminated water and soil, and direct exposure to the urine of infected animals which may lead to potential lethal disease (Ahmed, et al., 2006; Hermann-Storck et al., 2010; Villanueva et al., 2010; Zakeri, et al., 2010). The early rapid and accurate diagnosis of infections caused by pathogens is of utmost importance in the management of infectious diseases such as dengue, tuberculosis, typhoid fever, leptospirosis and schistosomiasis. In the Philippines, for instance, the most commonly used tests available in laboratories of large medical centers for definitive dengue diagnosis are the dengue antibody tests and the dengue NS1 antigen capture ELISA tests. Nucleic acid amplification such as the polymerase chain reaction (PCR) is promising but expensive. Further, it requires highly sophisticated equipments and may not be applicable in the field particularly in certain outbreaks.

In the case of tuberculosis, diagnostics and therapeutic management have always been emphasized as key to combat the disease but the same have remained to be major challenges in the fight against TB. Current tests to detect MTB rely heavily on microscopic observation and mycobacteria cultivation. The high sensitivity and specificity of the bacterial culture method remains the gold standard in detecting MTB. However, this technique is labor-intensive and time-consuming requiring 6-8 weeks for MTB to grow. The microscopic observation of acid-fast bacilli produces rapid results but has limitations in sensitivity (20% - 80%, Steingart et al, 2007) and specificity (Pandey et al., 2008). In addition, it is highly insensitive for extrapulmonary specimens, HlV-coinfected individuals and for children attributable to low bacillary loads in these patients (Getahun et al, 2007). Grave consequences of continuous transmission and death before management emphasize the need for rapid diagnostic methods for TB and for more timely treatment.

Presently, molecular methods, such as PCR, line probe assays and available commercial kits for TB detection, are gaining popularity at par with traditional TB diagnostic techniques. A major concern though is the need for costly equipment and trained personnel for molecular assays to be utilized regularly in clinical mycobacteriology laboratories especially in developing countries (Palomino, 2005). Most methods available for detecting Salmonella such as blood culture, Widal test, Typhidot,

Typhidot M, Tubex and PCR techniques lack the simplicity and efficiency of a good diagnostic kit as well as speed in detection. In the Philippines, the use of culture-based techniques is still being performed in diagnostic laboratories. This method provides the direct detection of Salmonella infection. However, this method requires the use of equipment and properly trained personnel. The turnaround time for the results of culture-based techniques is at least 24 hours for positive detection. Serological tests such as TUBEX and Typhidot have been shown to have a short turnaround time for results. These tests were evaluated by Kawano et al (2007) in the Philippines and showed that TUBEX has 94.7% sensitivity and 80.4% specificity, Typhidot IgM has 54.7% sensitivity and 64.7% specificity, Typhidot IgG has 73.3% sensitivity and 46.1% specificity while SD bioline IgM has 69% sensitivity and 79.3 specificity. The four serological methods are limited due to possible cross reaction that would result to false positive results. Leptospirosis has the greatest impact on health and economy in developing countries where it is often under-recognized and has affected livestock animals. The Philippines also faces a serious problem of Leptospirosis affliction. Poor sanitation and the increase of population in urban slums along with frequent typhoons and expansion of flooding areas in the country have increased the risk of Leptospira infection (Victoriano et al., 2009; Zakeri et al., 2010; Sonthayanon et al., 2011).

Before 1989, members of the genus Leptospira are conventionally grouped into two separate species based on their pathogenecity: the parasitic Interrogans Group which are pathogenic and are normally isolated from the patient's blood, urine and cerebrospinal fluid; and the saprophytic biflexa group which the nonpathogenic species. The serovar classification of Leptospira is based on the expression of the surface-exposed epitopes in a mosaic of the lipopolysaccharide (LPS) antigens, while the specificity of epitopes depends on the sugar composition and orientation (Adler and Moctezuma, 2010; Zakeri et al., 2010). Recently, genetic methods have attempted to replace the traditional classification methods and DNA-DNA hybridization studies were able to identify 20 Leptospira species (Cerqueira and Picardeau, 2009).

With the foregoing challenges in the detection of pathogens, the LAMP assay provides a powerful gene amplification technique for rapid identification of microbial infections. This method is the subject of EP 1020534. The LAMP assay was also described by Notomi, et al. (2000) (Loop- mediated isothermal amplification of DNA, Nucleic Acids Research, 28:e63 (2000)) which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. As compared to the widely used PCR-based detection, the LAMP assays also offer greater speed in obtaining results and less susceptibility to inhibitory substances Kaneko, et al. (2007). Nevertheless, the use of the LAMP assay is still a complicated process requiring a specific level of expertise on the part of the person performing the same. Further, the LAMP assay still has to be conducted in a laboratory with the proper equipment. Especially in developing countries, where tropical diseases are more prevalent, there is a need to provide a method and kit for rapid detection of pathogens that an ordinary health worker can use and/or perform even outside the laboratory.

Summary of the Invention

One embodiment of the invention provides a point-of-care testing through the use of portable instruments or diagnostic kits.

According to one embodiment of the invention, there is provided a method for the detection of a pathogen in a sample by LAMP wherein the said sample is added to a reaction tube comprising of a theromostable reaction mixture and primers, incubating the reaction tube in a heater, and detecting the presence of pathogen in the sample. The pathogen that may be detected includes but is not limited to dengue, tuberculosis, typhoid fever, or leptospir sis. In another aspect of the embodiment of the invention, there is provided a single step detection of pathogens.

The above-described methods may be conducted outside the laboratory and even by trained ordinary health workers.

According to another embodiment of the invention, there is provided a kit for the detection of a pathogen in a sample by LAMP, comprising of primers and a thermostable reaction mixture.

In still another embodiment of the invention, there is provided a kit comprising of primers and a thermostable reaction mixture for a single-step detection of a pathogen by LAMP, by adding a sample to the thermostable reaction mixture and primers. The thermostable reaction mixture may be in a lyophilized form, in which case water will be added aside from the sample.

According to one embodiment of the invention, there is provided a kit comprising of primers and a thermostable reaction mixture that will simultaneously detect and serotype dengue virus infections using LAMP assay.

According to one embodiment of the invention, there is provided a kit comprising of primers and a thermostable reaction mixture that will discriminate between MTB and MOTT using LAMP assay.

According to one embodiment of the invention, there is provided a kit comprising of primers and a thermostable reaction mixture that will detect and discriminate typhoidal and non-typhoidal serovars of Salmonella enterica using LAMP assay.

According to one embodiment of the invention, there is provided a kit comprising of primers and a thermostable reaction mixture that will detect leptospira infections using LAMP assay.

The kits described above may be portable. Thus, they may be brought to localities heavily afflicted with diseases to provide early detection of pathogens even if there is no laboratory facility nearby. Another embodiment of the invention provides a thermostable reaction mixture for use in LAMP assay that would detect pathogens upon addition of a sample and primers. The thermostable reaction mixture may be in liquid or lyophilized format. Still another aspect of the invention provides several sets of novel primers for use in the detection of various pathogens in samples by LAMP assay.

Detailed Description of the Invention The following description is non-limiting and will discuss only aspects and embodiments of the present invention in more detail.

One embodiment of the present invention provides for a detection of nucleic acid sequences, particularly, the products of nucleic acid amplifications. The invention is very useful in monitoring the formation of the target nucleic acid produced during amplification under isothermal conditions using LAMP method.

In one embodiment, the detection method utilizes a thermostable reaction mixture containing all reagents needed for the LAMP assay, placed in one reaction tube.

In one embodiment, single-step method of detection is done by (i) adding water and the sample bearing the DNA/RNA template to the tube containing the lyophilized thermostable reagent mixture with the target-specific primers. Incubation in a heater for 60 minutes is done thereafter. Following incubation, a colorimetric detection is done by ocular inspection of the change in color. A positive reaction will have a yellow green color while a negative reaction will maintain its orange color; or (ii) the sample bearing the DNA/RNA template is added directly to the tube containing the thermostable reaction mixture in liquid format and incubation in the heater for 60 minutes. Addition of detection dye is done following incubation and color change is observed directly. One embodiment of the present invention utilizes a portable heater that provides a temperature range of 65-67°C to achieve the isothermal condition required by a LAMP procedure.

Example 1. Dengue In one embodiment of the present invention, the pathogen that may be detected is the dengue virus. This embodiment of the invention involves the use of a thermostable reaction mixture that would detect only, or simultaneously detect and at the same time serotype dengue virus infections from a human sample which may contain RNA virus through the single-step detection method described above, i.e. :

(i) adding water and the sample bearing RNA template to the tube

containing the lyophilized thermostable reagent mixture with the target- specific primers. Incubation in a heater for 60 minutes is done thereafter.

Following incubation, a colorimetric detection is done by ocular inspection of the change in color. A positive reaction will have a yellow green color while a negative reaction will maintain its orange color; or

(ii) the sample bearing the RNA template is added directly to the tube

containing the thermostable reaction mixture in liquid format and incubation in the heater for 60 minutes. Addition of detection dye is done following incubation and color change is observed directly.

In one embodiment of the invention, the samples may be heated for 60 minutes at 63-67 ^< in a heater.

According to one embodiment, the method for obtaining RNA from plasma sample may be done by eluting RNA from plasma samples stored in FTA cards. The reaction mixture may be in a dry format containing (0.48-1.6 uM each of FLP and BIP; 0.24-0.8 uM each of F2 and B2; 0.2 uM each of F3 and B3; 1.4 mM deoxynucleoside triphosphates (dNTPs); 0.8 M betaine; 0.1% Tween 20; 10 mM (NH4)2S04; 8 mM MgSO; 10 mM KC1; 20mM TrisHCl pH 8.8; 0.64 U of Bst DNA polymerase, 0.0225-0.03 U of prepared Thermoscript or AMV reverse transcriptase). The thermostable reaction mixture may be in liquid or lyophilized format.

Another embodiment of the present invention is the design of novel dengue oligonucleotide primers used for detecting the target RNA. In this embodiment, a combination is provided in a medium. The combination comprises:

(i) sequences of the CprM gene junction of the dengue virus that were

aligned and the consensus sequences were used to design LAMP primers that would capture all four serotypes. These constitute the novel dengue universal LAMP primers containing nucleic acid sequences of SEQ ID NOS. 25-30 (see Table 1 below) used to determine if a sample is positive or negative for the dengue virus; and

(ii) the serotype-specific oligonucleotide primers that are used for RT-LAMP assay amplification of dengue viruses. These constitute the novel dengue serotype-specific LAMP primers containing nucleic acid sequences of SEQ ID NO. 1-6 for serotype 1, SEQ ID NO. 7-12 for serotype 2, SEQ ID NO. 13-18 for serotype 3 and SEQ ID NO. 19-24 for serotype 4 are used to identify the specific serotype of the dengue virus.

The foregoing primers were designed from the CprM gene junction of the Dengue Virus. The nucleotide sequences of the prototype strains of each dengue virus serotype were from 2008-2010 Philippine Dengue isolates. Potential target regions were selected from the aligned sequences, and RT-LAMP assay primers were designed for each serotype. A set of six primers comprising two outer (F3-Forward outer primer; B3- Backward outer primer), two inner (FIP- Forward Inner Primer; BIP- Backward Inner Primer), and two loop primers (F2-Forward loop primer; B2- Backward loop primer) that recognize eight distinct regions on the target sequence was designed. The primers were selected based on the criteria described by Notomi et al. (2000). After the primer design, many factors have to be considered such as the stability of the primers, avoiding primer-dimers or self-complementarity and the melting temperatures. Once primers were generated, this does not mean that it can be used directly in the LAMP assay. The above mentioned conditions must be determined prior to use the primers in the assay.

The primers are listed in Table 1 below:

11 DENV2-F2 AGATTGGAAGGATGCTGAA

12 DENV2-B2 CTTTTCCCTTTCTCTTGTCTAC

Dengue Serotype 3 Specific Primers

13 DENV3-F3 GGCCAAGGACCAATGAAACT

14 DENV3-B3 AGCGATGTCTTTTTCCGTCT

15 CCACCTAGCCAAAACTCCTGCTGGCGTTCATAGCTTTCCTC

DENV3-FIP A

16 GTACCTTCAAGAAGTCGGGGGCATGATGCTCAGCATGTTCG

DENV3-BIP A

17 DENV3-F2 GGCGTTCATAGCTTTCCTCA

18 DENV3-B2 ATGATGCTCAGCATGTTCGA

Dengue Serotype 4 Specific Primers

19 DENV3-F3 CTTTGCGGATGGTGCTAG

20 DENV3-B3 GTGGGAATCAAACACAGC

21 TCCCCATCTTTTCAGAATCCCTG-

DENV3-FIP CATTCATCACGTTTTTGCGA

22 AAGAACAAGGCCATCAAAATACTGACTTCTTCTCCTATTCA

DENV3-BIP AGATGTT

23 DENV3-F2 CATTCATCACGTTTTTGCGA

24 DENV3-B2 CTTCTTCTCCTATTCAAGATGTT

Dengue Universal Primers

25 UDENV-F3 TTAAAAAGATGGGGAACAATCA

26 UDENV-B3 CCCTTTCTCTTGTCTACTAACG

27 CCTGTTCAAGATGTTCAGCATCCTAAGGCTATCAATGTCTT

UDENV-FIP GAGA

28 UDENV-BIP GCAGAACTGCAGGTGTGATCATATGTGTGGTTCTCCGTTG

29 UDE V-F2 AAGGCTATCAATGTCTTGAGA

30 UDENV-B2 ATGTGTGGTTCTCCGTTG

Another embodiment of the invention provides a kit to detect the presence of dengue infections in patients with symptoms associated with dengue fever. The kit is a single-step detection system wherein a reaction is achieved by just adding a sample to a thermostable reaction mixture and primers, or additionally adding if the thermostable reaction mixture is in a lyophilized format.

In another embodiment of the invention, the kit may consist of: (a) the Universal Dengue Kit, which detects the presence of dengue virus in the sample; and/or (b) the Serotype-Specific Dengue Kit, which detects the specific serotype of dengue virus in the sample. Both kits contain thermostable reaction mixes in either liquid or dry format.

Another embodiment of the invention is a thermostable reaction mixture in a single reaction tube wherein a detection is achieved in two ways (i) addition of RNA template to the liquid format of the thermostable reaction mixture, incubation in a heater and addition of detection dye; or (ii) just by adding water and the RNA to the lyophilized thermostable reaction mixture, incubation in the heater and ocular inspection of the color change. Another aspect of the present invention is the method of extraction of the RNA from samples.

In this aspect, a combination is provided in a medium. The combination comprises (i) a plasma sample suspected of containing the target RNA virus, the target polynucleotide being in single stranded form (ii) Storing the plasma sample in the FTA cards capable of storing and preserving the RNA at room temperature (iii) purification of the RNA from the FTA cards (iv) elution of the RNA from the FTA card by using an RNA Processing Buffer. The buffer contains lOmM Tris-HCl, pH 8.0, 0.1 mM EDTA, 800U/mL RNase inhibitor and 2mM DTT.

Another embodiment of the present invention is a kit for use in amplification and detection of a target RNA. The kit is a packaged combination and comprises predetermined amounts of reagents, including the primers for conducting an amplification of the target RNA. The RT-LAMP reaction was tested out in a total 25-μ1 reaction mixture with the following range concentration for each of the following components: 0.48-1.6 μΜ each of F1P and BIP; 0.24-0.8 μΜ each of F2 and B2; 0.2 μΜ each of F3 and B3; 1.4 mM deoxynucleoside triphosphates (dNTPs); 0.8 M betaine; 0.1% Tween 20; 10 mM (NH4)₂S04; 8 mM MgS0₄; 10 mM KC1; 20 mM Tris-HCl pH 8.8; 0.64 U of Bst DNA polymerase, 0.0225-0.03 U of prepared ThermoScript Reverse Transcriptase or avian myeloblastosis virus reverse transcriptase (Invitrogen), and the 3μ1 of target RNA, which was incubated at 63-67°C for 60 min in a heater.

The analysis of each patient blood sample will be performed in a set of three tubes for Universal Dengue Kit (Positive Control, No Template Control, and sample). Each tube will contain all the LAMP primers for dengue. For the Serotype-Specific Dengue Kit, analysis will be done in a set of six tubes each containing the primer mixture for a particular serotype. Positive and negative controls will also be included in each run, and all precautions to prevent cross-contamination will be observed. Positive results will have a change in color from orange to yellow green.

Another aspect of the present invention is final format of a kit for the amplification of the target RNA. The kit comprises in packaged combination of the reaction mix, the Bst DNA polymerase, reverse transcriptase and the primers. The kit will be distributed in a lyophilized or dried format. Different protocols for the optimization of the lyophilization procedure were done including: (1) lyophilization of the RT-LAMP mastermix with trehalose as the stabilizer as compared to the setup without stabilizers, (2) lyophilization of the mastermix excluding the enzymes, and (3) lyophilization of the individual components of the RT- LAMP mixture.

Table 2. The table below shows the concentration of the components of the Thermostable Reaction Mix in lyophilized format.

The kit in lyophilized format may be stored at room temperature. The recommended protocol is as follows: remove the kit from the storage area. Dispense 22μ1 of water to each tube containing the thermostable reaction mixture and add 3 μΐ RNA sample or water for the NTC (No Template Control) or Positive Control. Reagents are mixed by tapping the tubes. After which, tubes are incubated in a heater. After incubation, results are determined colorometrically.

Table 3. The table below shows the concentration of the components of the Thermostable Reaction Mix in liquid format. Reagent Kit for Dengue

(Final Concentration)

Forward Inner Primer FIP= 0.48 - 1.6 μΜ

Backward Inner Primer BIP= 0.48 - 1.6 μΜ

Forward Outer Primer F3= 0.2 μΜ

Backward Outer Primer B3=0.2 μΜ

Forward Loop Primer F2= 0.24 - 0.8 μΜ

Backward Loop Primer B2=0.24-0.8 μΜ

lOx Reaction Buffer containing: 1 X

Betaine 0.8 M

dNTPs 1.4 mM each

MgSo4 14 mM total

Bst DNA Polymerase 0.64 U

Reverse Transcriptase AMV/Thermoscript Reverse Transcriptase = 0.0225 - 0.03

U

Nucleic Acid Stain 37 X (SYBR Green I)

The kit in liquid format may be stored either at -20 C. The recommended protocol is as follows: remove the kit from the refrigerator or freezer. When the kit is stored in the freezer, allow to thaw the reaction mix. Once thawed, put 22μ1 of the thermostable reaction mixture per PCR tube and add 3 μΐ RNA sample or water for the NTC (No Template Control) or Positive Control. Reagents are mixed by tapping the tubes. After which, tubes are incubated in a heater. After incubation, addition of 2 μΐ detection dye is done to determine results colorometrically.

Sensitivity and Specificity

The Universal Dengue Kit and the Serotype-Specific Dengue Kit were compared with in- house heminested RT-PCR assay as the gold standard.

The RT-PCR was carried out in a 25-ul reaction volume using published primers: mDl, rTSl, mTS2, TS3, and mTS4 for the serotyping of dengue. Detection of PCR product was then carried out in a 1.5% Agarose Gel and subsequent serotyping of dengue was done basing on the corresponding amplicon size per serotype.

Sensitivity and Specificity Assay of the Universal Dengue Kit A total of 105 samples were used to assess the over-all sensitivity of the Universal Dengue Kit (indicated as RT-LAMP in Table 4 below). Seventy nine samples were from confirmed cases of Dengue and 26 samples were acute febrile sera with alternative diagnosis. Confirmed cases are defined as acute febrile sera testing positive for dengue by Hemi-nested RT-PCR and sequencing.

The overall sensitivity of the Universal Dengue Kit Assay is 95% and the Specificity is 100%. Its Negative Predictive Value (NPV) is 86.6% and its Positive Predictive Value (PPV) is 100%.

Table 4. Sensitivity testing of the Universal Dengue Kit (n=105)

Serotype Discriminatory Power Testing

The analysis of each sample was performed in a set of five tubes, each of which had the primer mixture for a particular serotype and for the universal primer mixture. Positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed. Positive for dengue virus infection should have a change in color for the Udenv primers and in another tube containing a specific serotype.

Serotype Discriminatory Power for Serotype 1

A total of 28 samples with confirmed dengue 1 cases from a total of 61 cases presenting with acute febrile illness. The sensitivity of the Serotype 1 -Specific RT-LAMP was calculated to be 78%, Specificity is 48%, Negative Predictive Value (NPV) is 72% and Positive Predictive Value (PPV) is 56%.

Table 5. Sensitivity Testing of the Serotype 1 -Specific RT-LAMP Assay (n=61)

-

L (+) 22 17 39

A

M (-) 6 16 22

P

Total 28 33 61

Serotype Discriminatory Power for Serotype 2

A total of 24 samples were confirmed dengue 2 cases from a total of 32 cases presenting with acute febrile illness. The sensitivity of the Serotype 2-Specific RT-LAMP was calculated to be 91.66%, Specificity is 100%, Negative Predictive Value (NPV) is 80% and Positive Predictive Value (PPV) is 100%.

Table 6. Sensitivity Testing of the Serotype 2-Specific RT-LAMP Assay (n=32)

Serotype Discriminatory Power for Serotype 3

A total of 19 samples were confirmed dengue 3 cases from a total of 81 cases presenting with acute febrile illness. The sensitivity of the Serotype 3-Specific RT-LAMP was calculated to 84%, Specificity is 82%, Negative Predictive Value (NPV) is 94% and Positive Predictive Value (PPV) is 59%.

Table 7. Sensitivity Testing of the Serotype 3-Specific RT-LAMP Assay (n=81)

L (+) 16 11 27

A

M (-) 3 51 54

P

Total 19 62 81

Serotype Discriminatory power for Serotype 4

A total of 3 samples were confirmed dengue 4 cases from a total of 44 cases presenting with acute febrile illness. The sensitivity of the Serotype 4-Specific RT-LAMP was calculated to 100%, Specificity is 85.4%, Negative Predictive Value (NPV) is 100% and Positive Predictive Value (PPV) is 33.3%.

Table 8. Sensitivity Testing of the Serotype 4-Specific RT-LAMP Assay (n=44)

The universal primer was designed using the individual primers of the different serotypes. The results for detection was therefore better than the individual serotype assays since its primer was able to bind to a broader target relative to the individual ones; that is why the term universal was used since it can and should capture all serotypes. Cross reactivity of some of the serotype specific primers have been observed to occur; this may be due to the fact that they have sequences that are overlapping within the primers.

Example 2. Leptospirosis In another aspect of the present invention, the pathogen that may be detected is Leptospira.

One embodiment of the present invention is a method for the detection of pathogenic Leptospira species in human samples including blood, urine and cerebrospinal fluid (CSF), targeting the ompLl gene.

Another aspect of the present invention is the design of the novel oligonucleotide leptospirosis LAMP primers used for detecting the target R A. In this embodiment, a combination is 5 provided in a medium. The combination comprises sequence primers designed from the ompLl gene.

The target gene is found to be present only in the pathogenic species of the Leptospira species. Potential target regions were selected from the aligned sequences, and primers were designed. A set of six primers comprising two outer (F3-Forward outer primer; B3- Backward outer primer), two inner (FIP- Forward Inner Primer; BIP- Backward Inner Primer), and two loop primers (F2-Forward loop 10 primer; B2- Backward loop primer) that recognize eight distinct regions on the target sequence was designed. The primers were selected based on the criteria described by Notomi et al. (2000).

The primers are listed in Table 9 below:

5 Another embodiment of the present invention is a kit for use in amplification and detection of a target Leptospira DNA. The kit is a packaged combination and comprises predetermined amounts of reagents, including the primers for conducting an amplification of the target DNA. The LAMP reaction was tested out in a total 25-μ1 reaction mixture with the following range concentration for each of the following components: 0.48-1.6 μΜ each of FIP and BIP; 0.24-0.8 μΜ each of F2 and B2; 0 0.2 μΜ each of F3 and B3; 1.4 mM deoxynucleoside triphosphates (dNTPs); 0.8 M betaine; 0.1% Tween 20; 10 mM (NH4)₂S04; 8 mM MgS0₄; 10 mM KC1; 20 mM Tris-HCl pH 8.8; 0.64 U of Bst DNA polymerase, and the 4μ1 of target DNA, which was incubated at 63-67°C for 60 min in a heater.

Another embodiment of the invention is the thermostable reaction mixture in a single reaction tube wherein a detection is achieved in two ways (i) addition of DNA template to the liquid format of the thermostable reaction mixture, incubation in a heater and addition of detection dye (ii) just by adding water and the DNA to the lyophilized thermostable reaction mixture, incubation in a heater and ocular inspection of the color change.

Specificity and Sensitivity

Specificity and sensitivity were used as measures of accuracy. The PPV and the NPV were likewise obtained. The specificity of the assay was evaluated by using serum samples from patients with acute febrile illness.

To compare the sensitivity of the Leptospirosis Kit to that of the PCR assay, the same batch of bacterial DNA extracted from the culture of reference standards and serum samples from patients with either confirmed leptospira infection or serum samples from patients with acute febrile illness were used for PCR assay. The reactions were carried out in a 25 μΐ volume containing 5 μΐ of the extracted DNA, dNTPs, Titanium Taq (Clontech), 25 mM MgCl₂ and 5x Reaction Buffer. Each reaction mixture was subjected to 30 cycles of amplification (95°C for 15 sec, 51°C for 15 sec and 72°C for 45 sec) and a final extension at 72°C for 5 minutes. QLAGEN DNeasy Blood and Tissue kit was used for genomic DNA extraction and followed according to the manufacturer's instruction.

A total of 38 samples (12 were confirmed cases, 21 were serum samples obtained from patients with acute febrile illness and 5 were from reference cultures of Leptospira serovars) were tested for the Leptospirosis Kit. Sensitivity was calculated to be 92%, Specificity is 88%, NPV is 96% and PPV is 76%.

Example 3. TB In another aspect of the present invention, the pathogen that may be detected is

Mycobacterium tuberculosis.

One embodiment of the present invention is a method for the detection and discrimination of MTB and MOTT through the single-step detection method described above.

Another embodiment of the present invention is the design of novel oligonucleotide primers used for detecting the target DNA. Sequences of the hsp65 and rpoB gene of MTB and select members of MOTT (eg. M. avium, M. intracellulare, M. gordonae, M. fortuitum, M. abscessus, M. smegmatis, M. marinum, M. terrae, M. massiliense) were aligned and the variant portions of the gene between the two groups of mycobacteria were used to design the LAMP primers. A set of six primers comprising two outer (F3-Forward outer primer; B3- Backward outer primer), two inner (FIP- Forward Inner Primer; BIP- Backward Inner Primer), and two loop primers (F2-Forward loop primer; B2- Backward loop primer) that recognize eight distinct regions on the target sequence was designed. The primers were selected based on the criteria described by Notomi et al (2000). After the primer design, various factors have to be considered, such as, the stability of the primers, its tendency to form primer-dimers or self-complementarity and the melting temperatures. Once the primers are generated, they cannot be used directly in the LAMP assay. The above mentioned conditions must be determined and assessed prior to use of the primers in the assay.

Table 10. Primers designed for the detection of MTB based on hsp65 and rpoB gene.

G

TB- hsp65-F3-D TCCGGCGTCTTGCGT

TB- hsp65-B3-D GGTCGCCGGAGACTTCAC

TB- hsp65-F2-D GTGCTCGTCGCGGTGTT

TB- hs_P65-B2-D GCCGAGATCGTCAAGGATG

TB- hsp65-FIP-E AGACGCCGGAGACAAAGACGGGGAACGAGCGGCGGAA

TB- hsp65-BIP-E GATCACCAGGCGGCTGACCGAACTGCCCGGCATTGA

TB- hsp65-F3-E GCCTCGCTGGTGACGT

TB- hsp65-B3-E GTCAAGGATGGCGACGAC

TB- hs_P65-F2-E GGAACGAGCGGCGGAA

TB- hs_P65-B2-E GAACTGCCCGGCATTGA

TB-rpoB-F3-A CATCTTCGGTGAGAAGGCC

TB-rpoB-B3-A CTTGTCACCGTCGGAGATC

TB-rpoB-FIP-A AATGCCGATCACCTTGCCGGACGAGGTGCGCGACACT

TB-rpoB-BIP-A GTGTTTTCCCGCGAGGACGATTGCGTTTCTGAGCCACAT

TB-rpoB-F2-A CGAGGTGCGCGACACT

ΤΒ- οΒ-Β2-Α TTGCGTTTCTGAGCCACAT

ΤΒ-φοΒ-Ρ3-Β GCCGGTGTCAACGAGCT

ΤΒ-φθΒ-Β3-Β CGATGTTCATCCGTCGCG

ΤΒ-φοΒ-FIP-B TCACGCCCTTGTTGCCGTGGTGCGTGTGTATGTGGCT

ΤΒ-φοΒ-ΒΙΡ-Β GATCCTGCCGGTTGAGGACATACGCCGTGGGTGTTCAA

ΤΒ-φοΒ-Ρ2-Β GTGCGTGTGTATGTGGCT

ΤΒ-φοΒ-Β2-Β ACGCCGTGGGTGTTCAA

TB^oB-F3-C GCTGCCCGACGAACTG

ΤΒ-φοΒ-Β3-0 ACCGGGTACGGGAACG

ΤΒ-φοΒ-FIP-C AACAGGCCCTGCAGCTCGCGCAGCCGAACGCCAT

ΤΒ-φοΒ-ΒΙΡ-C GTCGTGCACGCTGCCCAATGCGCCCGTCGAAGAG

TB^oB-F2-C CGCAGCCGAACGCCAT

ΤΒ-φοΒ-Β2-0 TGCGCCCGTCGAAGAG

TB^oB-F3-D TGTATGTGGCTCAGAAACGC

ΤΒ-φοΒ-Β3-ϋ CCCAGGTGGGTCTCCAA

ΤΒ-φοΒ-FIP-D AGGATCTTGCCGATCACGCCCAAGATCTCCGACGGTGACA

ΤΒ-φοΒ-ΒΙΡ-D GTTCCTTGCCGACGGCACCCTGGCCGATGTTCATCCG

ΤΒ-φοΒ-Ρ2-ϋ AAGATCTCCGACGGTGACA

ΤΒ-φοΒ-Β2-0 CTGGCCGATGTTCATCCG 91 TB-rpoB-F3-E CGAGGACGAGGACGAGTTG

92 TB-rpoB-B3-E ACGCCGTGGGTGTTCAA

93 TB-rpoB-FIP-E GCCAGCTTGTCACCGTCGGAGCAACGAGCTGGTGCGTGT

94 ΤΒ- οΒ-ΒΙΡ-Ε GCAACAAGGGCGTGATCGGCGCCGTCGGCAAGGAAC

95 ΤΒ-φοΒ-Ρ2-Ε CAACGAGCTGGTGCGTGT

96 ΤΒ-φοΒ-Β2-Ε GCCGTCGGCAAGGAAC

Table 11. Primers designed for the detection of Mycobacterium Other Than Tuberculosis (MOTT) based on hsp65 and rpoB gene.

120 MOTT - hsp65-B2-D GCCTCGGCGATCAGGT

121 MOTT - hs_P65-F3-E GCCTCGGCGATCAGGT

122 MOTT - hsp65-B3-E TGTCGAACCGCATACCCTC

123 MOTT - hsp65-FIP-E CAGGTCGCCGATCGACTGGTCAAGAGGTCGAGACCAAGGAC

124 MOTT - hsp65-BIP-E CGAGGCGATGGACAAGGTCGCGAGCTGCAGGCCGAA

125 MOTT - hs_P65-F2-E AAGAGGTCGAGACCAAGGAC

126 MOTT - hs_P65-B2-E CGAGCTGCAGGCCGAA

127 MOTT-rpoB-F3 -A GACGGCGACAAGCTGG

128 MOTT-rpoB-B3-A CACCCGAGGTGGGTCT

129 MOTT-rpoB-FIP-A AGGAACGGCATGTCCTCCTGGGGACGGCACGGCAACA

130 MOTT-rpoB-BIP-A GGCACTCCGGTGGACATCATAGGATCTGGCCGATGTTCAT

131 MOTT-rpoB-F2-A GGACGGCACGGCAACA

132 MOTT-rpoB-B2-A AGGATCTGGCCGATGTTCAT

133 MOTT-rpoB-F3-B GTCCGGCTGGAACATCG

134 MOTT-rpoB-B3-B TGCGGCCGTCGAACAG

135 MOTT-rpoB-FIP-B ACGATCTGGTCCGGCTGCGCCCGATTGGGCGGTGAA

136 MOTT-rpoB-BIP-B GCGCCAAGGAGGAGGAGCTTCGCCGTCCACCATGAC

137 MOTT-rpoB-F2-B CCCGATTGGGCGGTGAA

138 MOTT-rpoB-B2-B TCGCCGTCCACCATGAC

139 MOTT-rpoB-F3-C GCTGAAGGTGCCGCAC

140 ΜΟΤΤ- οΒ-Β3-0 GAACGGCATGTCCTCCTG

141 MOTT-rpoB-FIP-C ACCAGCTCGTTGACACCGGCGAGTCCGGCAAGGTCATCG

142 ΜΟΤΤ-φθΒ-ΒΙΡ-C CGCGTCTACGTGGCCCAGAAGGCCGATCACACCCTTGTTG

143 MOTT-4-poB-F2-C GAGTCCGGCAAGGTCATCG

144 MOTT-rpoB-B2-C GCCGATCACACCCTTGTTG

145 MOTT^oB-F3-D TGGGTCGCCAAGTCCG

146 ΜΟΤΤ-φθΒ-Β3-ϋ AACAGCACCGACTTGCC

147 ΜΟΤΤ-φθΒ-FIP-D ACGATCTGGTCCGGCTGCGATCGAGGGCTCACCCGAT

148 ΜΟΤΤ-φθΒ-ΒΙΡ-D GCGCCAAGGAGGAGGAGCT-TCGCCGTCCACCATGAC

149 MOTT^oB-F2-D ATCGAGGGCTCACCCGAT

150 ΜΟΤΤ-φθΒ-Β2-ϋ TCGCCGTCCACCATGAC

151 ΜΟΤΤ-φθΒ-Ή-Ε CGGCCGGTGTCAACGA

152 ΜΟΤΤ-φθΒ-Β3-Ε CCGATGTTCATCCGTCGC

153 ΜΟΤΤ-φθΒ-FIP-E TTGTTGCCGTGCCGTCCGGTGCGCGTCTACGTGGC

154 ΜΟΤΤ-φθΒ-ΒΙΡ-Ε GCAAGATCCTGCCCCAGGAGACCCCGTGGGTGTTCAG 155 MOTT-rpoB-F2-E TGCGCGTCTACGTGGC

156 MOTT-rpoB-B2-E ACCCCGTGGGTGTTCAG

Another embodiment of the present invention is a kit for use in amplification and detection of a target DNA. The kit is a packaged combination and comprises predetermined amounts of reagents, including the primers for conducting an amplification of the target DNA. The LAMP reaction was tested out in a total of 25 -μΐ reaction mixture with the following range concentration for each of the following components: 0.48-1.6 μΜ each of FIP and BIP; 0.24-0.8 μΜ each of F2 and B2; 0.2 μΜ each of F3 and B3; 0.4 - 1.4 mM deoxynucleoside triphosphates (dNTPs); 0.8 - 1M betaine; 0.1 % Tween 20; 10 mM (NH4)₂S04; 6-8 mM MgS0₄; 10 mM KC1; 20 mM Tris-HCl pH 8.8; 0.32-0.64 U of Bst DNA polymerase and 3μ1 of target DNA, which was incubated at 63-67°C for 60 min in a heater. The analysis of each extracted DNA was performed in two sets of three tubes each: the MTB set (Positive MTB Control, No Template Control, and Sample) and the MOTT set (Positive MOTT Control, No Template Control, and Sample). Following incubation at 63-67°C for 60 min in a heater, LAMP amplicons were directly detected by adding 2μ1 of 1/20-diluted detection dye. The tubes with amplification (positive result) changed in color from orange to yellow green. Also, to confirm a positive LAMP reaction, 5-μ1 aliquot of the LAMP assay products were electrophoresed on 1.5% agarose gel in TAE buffer, followed by staining with ethidium bromide and visualization on a UV transilluminator to confirm results. In the field, it is not necessary to do agarose gel electrophoresis.

Another aspect of the present invention is the final format of a kit for the amplification of the target DNA. The kit comprises in packaged combination of the reaction mix, the Bst DNA polymerase and the primers. The kit will be distributed in a lyophilized or dried format. Different protocols for the optimization of the lyophilization procedure were done including: (1) lyophilization of the LAMP mastermix with trehalose as the stabilizer as compared to the set-up without stabilizers, (2) lyophilization of the mastermix excluding the enzymes, and (3) lyophilization of the individual components of the LAMP mixture.

Another embodiment of the invention is a thermostable reaction mixture in a single reaction tube wherein a detection is achieved in two ways (i) addition of DNA template to the liquid format of the thermostable reaction mixture, incubation in a heater and addition of detection dye ; or (ii) just by adding water and the DNA to the lyophilized thermostable reaction mixture, incubation in the heater and ocular inspection of the color change.

Table 12. The table below shows the concentration of the components of the Thermostable Reaction Mix in liquid format. Reagent Kit for TB

(Final Concentration)

Forward Inner Primer FIP= 0.48 - 1.6 μΜ

Backward Inner Primer BIP= 0.48 - 1.6 μΜ

Forward Outer Primer F3= 0.2 μΜ

Backward Outer Primer B3=0.2 μΜ

Forward Loop Primer F2= 0.24 - 0.8 μΜ

Backward Loop Primer B2=0.24-0.8 μΜ

1 Ox Reaction Buffer containing: 1 X

Betaine 0.8 - 1 M

dNTPs 0.4 - 1.4 mM

MgSo4 6 - 8 mM

Bst DNA Polymerase 0.32 - 0.64 U

Nucleic Acid Stain 37 X (SYBR Green I)

The TB Kit in liquid format may be stored either at -20°C. The recommended protocol is as follows: Remove the kit from the refrigerator or freezer. When the kit is stored in the freezer, thaw the reaction mix. Once thawed, put 22μ1 of the thermostable reaction mixture per PCR tube and add 3 μΐ DNA sample or water for the NTC (No Template Control) or Positive Control. Reagents are mixed by tapping the tubes. After which, tubes are incubated in a heater. After incubation, addition of 2 μΐ detection dye is done to determine results colorimetrically.

Table 13. The table below shows the concentration of the components of the Thermostable Reaction Mix in lyophilized format.

Bst DNA Polymerase 0.32 - 0.64 U

Nucleic Acid Stain 37 X (SYBR Green I)

Cryoprotectants/Lyoprotectants 0.2-0.4 M Dextrane/ Gelatine/

Trehalose/Saccharose/Glucose

The kit in lyophilized format may be stored at room temperature. The recommended protocol is as follows: Remove the kit from the storage area. Dispense 22μ1 of water to each tube containing the thermostable reaction mixture and add 3 μΐ DNA sample or water for the NTC (No Template Control) or Positive Control. Reagents are mixed by tapping the tubes. After which, tubes are incubated in a heater. After incubation, results are determined colorimetrically.

Specificity and Sensitivity Identification of MTB was done by observing characteristic colonial morphology and growth patterns of the mycobacterium in M/B BacT/7H9 broth and Lowenstein-Jensen (LJ) or in Ogawa medium for sixty-seven (67) heat-deactivated MTB isolates.

All sixty-seven (67) MTB isolates were tested for LAMP-PCR using MTB LAMP primers while a subset was tested for the Muniv LAMP primers. Sixty-six (66) of the tubes with MTB changed color from orange to light green when amplified using the MTB LAMP primers, while the tube without amplification remained orange. Only 1 out of 67 MTB isolates tested negative with the MTB LAMP primer demonstrating its potential use in frontline MTB diagnosis. For confirmation, the LAMP product was run through electrophoresis and found the pattern to be similar with that reported by Notomi et al. (2000). A successful LAMP reaction produces many bands while a reaction with no amplification produces no bands. The TB Kit demonstrates a 98.5% specificity and 100% sensitivity in detecting MTB using the LAMP protocol.

The Muniv LAMP primers were able to discriminate between mycobacterial and non- mycobacterial isolates. In this assay, the expected results for non-mycobacterium (NM), mycobacterium other than MTB (MOTT) and MTB is summarized in Table 15.

Table 14. Expected results from LAMP assay using Muniv and MTB primers.

(MOTT)

Mycobacterium tuberculosis Positive (L. green) Positive (L. green) complex (MTB)

Example 4. Typhoid

In another aspect of the present invention, the pathogen that may be detected is Salmonella enterica.

One embodiment of the present invention is a method for the detection and discrimination of typhoidal and non-typhoidal serovars of Salmonella enterica through the single-step detection method described above.

Another embodiment of the present invention is the design of the novel typhoid oligonucleotide primers used for detecting the target DNA. Sequences of the iroB gene of typhoidal and non-typhoidal serovars of Salmonella enterica were aligned and the consensus portions were used to design the universal Salmonella sp. LAMP primers. On the other hand, sequences of the sopE genes of both typhoidal and non-typhoidal serovars of Salmonella enterica were aligned and the variant portions between the two serogroups were used to design the LAMP primers specific only to the typhoidal serovars. A set of six primers comprising two outer (F3-Forward outer primer; B3- Backward outer primer), two inner (FIP- Forward Inner Primer; BIP- Backward Inner Primer), and two loop primers (F2-Forward loop primer; B2- Backward loop primer) that recognize eight distinct regions on the target sequence was designed. The primers were selected based on the criteria described by Notomi et al (2000). After the primer design, various factors have to be considered, such as, the stability of the primers, its tendency to form primer-dimers or self-complementarity and the melting temperatures. Once the primers are generated, they cannot be used directly in the LAMP assay. The above mentioned conditions must be determined and assessed prior to use of the primers in the assay.

Table 15. Primer sets for the universal detection of Salmonella enterica.

161 SAL-iroB-F2-A GGCACATCAAAGGCGTGA

162 SAL-iroB-B2-A CGCCGTTATACGGGACGT

163 SAL-iroB-F3-B GCCCTGGCACATCAAAGG

164 SAL-iroB-B3-B CGCTTGCGATCAGGTGTAC

165 SAL-iroB-FIP-B TGTGACGTCTATCCACGCCAGACACTTTCTAACGCCTACCGC

166 SAL-iroB-BIP-B CTGCAAAATGACGGAGAGCCGGGTTCCCACCATTCTTCCCAG

167 SAL-iroB-F2-B CACTTTCTAACGCCTACCGC

168 SAL-iroB-B2-B GTTCCCACCATTCTTCCCAG

169 SAL-iroB-F3-C TACGCCCTGGCACATCAA

170 SAL-iroB-B3-C ACGTTCCCACCATTCTTCC

171 SAL-iroB-FIP-C GATCTCTTGGTGGCGCGCTGGGCGTGACGAAATCACTTTC

172 SAL-iroB-BIP-C GCGTGGATAGACGTCACACCGCGGGACGTATTGCATGGAG

173 SAL-iroB-F2-C GGCGTGACGAAATCACTTTC

174 SAL-iroB-B2-C CGGGACGTATTGCATGGAG

175 SAL-iroB-F3-D GGCATTCCGCAGATAGTCTT

176 SAL-iroB-B3-D TGGGCCGCCATTTCCG

177 SAL-iroB-FIP-D TAATGCCGCATCCGCGCTCCCAGGGTGCCGACAGA

178 SAL-iroB-BIP-D TTCCCGGTAAGAGCGGGCTTACACCTCCTGCGACGCCT

179 SAL-iroB-F2-D CCAGGGTGCCGACAGA

180 SAL-iroB-B2-D ACCTCCTGCGACGCCT

181 SAL-iroB-F3-E TGGTCGAGCGCGGATG

182 SAL-iroB-B3-E TTAGCCGTGTTGCAGCAT

183 SAL-iroB-FIP-E AAGCGCGCGATTACCGAGGCCGGTAAGAGCGGGCTTA

184 SAL-iroB-BIP-E CAGGAGGTCGCGGCGGAAAGGCGATCAGTTTTTTTGCCA

185 SAL-iroB-F2-E CCGGTAAGAGCGGGCTTA

186 SAL-iroB-B2-E GGCGATCAGTTTTTTTGCCA

187 SAL-iroB-F3-F GACAGACCCGTCAATGCC

188 SAL-iroB-B3-F TTAGCCGTGTTGCAGCAT

189 SAL-iroB-FIP-F GCGCGATTACCGAGGAAGGTATTGGTCGAGCGCGGAT

190 SAL-iroB-BIP-F CAGGAGGTCGCGGCGGAAAGGCGATCAGTTTTTTTGCCA

191 SAL-iroB-F2-F TGGTCGAGCGCGGAT

192 SAL-iroB-B2-F GGCGATCAGTTTTTTTGCCA

Table 16. Primer sets for the detection of the typhoidal serovars of Salmonella enterica.

SEQ ID Primer Name Sequence 5' -> 3' NO.

193 TYPH-sopE-F3-A GCAGTGTTGACAAATAAAGTC

194 TYPH-sopE-B3-A TGTTGATCCCTTTGCTGA

195 TYPH-sopE-FIP-A GGTCTTTACTCGCACTACCTCTAAGTTAAAGATTTTATGCTT

CAAACGC

196 TYPH-sopE-BIP-A AGCCAGACCCGTGAAGCTATTGAGCAAATTACAACACTGAT

C

197 TYPH-sopE-F2-A GTTAAAGATTTTATGCTTCAAACGC

198 TYPH-sopE-B2-A TGAGCAAATTACAACACTGATC

199 TYPH-sopE-F3-B ACCGAGAAAAATTCTTTAGCAA

200 TYPH-sopE-B3-B CTCTAATATCTATATCATTGAGCGT

201 TYPH-sopE-FIP-B AGACTCAGTGTTCTTATGCGAAATAAAGTATTCTCGCAGTA

AAAAATCAC

202 TYPH-sopE-BIP-B GCAACACACTTTCACCGAGG- TGAAGCATAAAATCTTTAACGAC

203 TYPH-sopE-F2-B AAGTATTCTCGCAGTAAAAAATCAC

204 TYPH-sopE-B2-B TGAAGCATAAAATCTTTAACGAC

205 TYPH-sopE-F3-C GCTTCAAACGCTCAATGA

206 TYPH-sopE-B3-C GCAGACCTGCATTTTTCG

207 TYPH-sopE-FIP-C ATAGCTTCACGGGTCTGGCTTATAGATATTAGAGGTAGTGC

GAGT

208 TYPH-sopE-BIP-C CGGCAGTTTACAGCAAGAATAAAGAATTTCCTGAAGAAAA

GGCG

209 TYPH-sopE-F2-C TATAGATATTAGAGGTAGTGCGAGT

210 TYPH-sopE-B2-C ATTTCCTGAAGAAAAGGCG

211 TYPH-sopE-F3-D AATTATCGGAACGTTTTATTTCG

212 TYPH-sopE-B3-D CACTGATCTTTATTCTTGCTGTA

213 TYPH-sopE-FIP-D GACTTTATTTGTCAACACTGCCCGAGAACACTGAGTCTTCT

GC

214 TYPH-sopE-BIP-D TTAGAGGTAGTGCGAGTAAAGACC- GCCGATAGTATAGCTTCACG

215 TYPH-sopE-F2-D AGAACACTGAGTCTTCTGC

216 TYPH-sopE-B2-D GCCGATAGTATAGCTTCACG

217 TYPH-sopE-F3-E TTATTTCGCATAAGAACACTGA

218 TYPH-sopE-B3-E CACTGATCTTTATTCTTGCTGTA

219 TYPH-sopE-FIP-E GACTTTATTTGTCAACACTGCCCGGTCTTCTGCAACACACTT TC

220 TYPH-sopE-BIP-E TTAGAGGTAGTGCGAGTAAAGACCGCCGATAGTATAGCTTC

ACG

221 TYPH-sopE-F2-E GTCTTCTGCAACACACTTTC

222 TYPH-sopE-B2-E GCCGATAGTATAGCTTCACG

Another embodiment of the present invention is a kit for use in amplification and detection of a target DNA. The kit is a packaged combination and comprises predetermined amounts of reagents, including the primers for conducting an amplification of the target DNA. The LAMP reaction was tested out in a total of 25-μ1 reaction mixture with the following range concentration for each of the following components: 0.48-1.6 μΜ each of FTP and BIP; 0.24-0.8 μΜ each of F2 and B2; 0.2 μΜ each of F3 and B3; 1.4 mM deoxynucleoside triphosphates (dNTPs); 0.8 - 1M betaine; 0.1% Tween 20; 10 mM (NH4)₂S04; 8 mM MgS0₄; 10 mM KC1; 20 mM Tris-HCl pH 8.8; 0.32 - 0.64 U of Bst DNA polymerase and 3μ1 of target DNA, which was incubated at 63-67°C for 60 min in a heater. The analysis of each extracted DNA was performed in 2 sets of three tubes each: (i) Universal Salmonella set (Positive Control, Sample, No Template Control) and (ii) Typhoidal serovar-specific set (Positive Control, Sample, No Template Control). Following incubation at 63-67°C for 60 min in a heater, LAMP amplicons were directly detected by adding 2μ1 of 1/20-diluted detection dye. The tubes with amplification (positive result) changed in color from orange to yellow green. Also, to confirm a positive LAMP reaction, 5-μ1 aliquot of the LAMP assay products were electrophoresed on 1.5% agarose gel in TAE buffer, followed by staining with ethidium bromide and visualization on a UV transilluminator to confirm results. In the field, it is not necessary to do agarose gel electrophoresis.

Another aspect of the present invention is the final format of the kit for the amplification of the target DNA. The kit comprises in packaged combination of the reaction mix, the Bst DNA polymerase and the primers. The kit will be distributed in a lyophilized or dried format. Different protocols for the optimization of the lyophilization procedure were done including: (1) lyophilization of the LAMP mastermix with trehalose as the stabilizer as compared to the set-up without stabilizers, (2) lyophilization of the mastermix excluding the enzymes, and (3) lyophilization of the individual components of the LAMP mixture.

Another embodiment of the invention is the thermostable reaction mixture in a single reaction tube wherein a detection is achieved in two ways (i) addition of DNA template to the liquid format of the thermostable reaction mixture, incubation in a heater, addition of detection dye and ocular inspection of the color change; or (ii) just by adding water and the DNA to the lyophilized thermostable reaction mixture, incubation in a heater and ocular inspection of the color change. Table 17. The table below shows the concentration of the components of the Thermostable Reaction Mix in liquid format.

The Typhoid Kit in liquid format may be stored either at -20 C. The recommended protocol is as follows: Remove the kit from the refrigerator or freezer. When the kit is stored in the freezer, thaw the reaction mix. Once thawed, put 22μ1 of the thermostable reaction mixture per PCR tube and add 3 μΐ DNA sample or water for the NTC (No Template Control) or Positive Control. Reagents are mixed by tapping the tubes. After which, tubes are incubated in a heater. After incubation, addition of 2 μΐ detection dye is done to determine results colorimetrically.

Table 18. The table below shows the concentration of the components of the Thermostable Reaction Mix in lyophilized format.

Betaine 0.8 - 1 M

dNTPs 1.4 mM

MgSo4 8 mM

Bst DNA Polymerase 0.32 - 0.64 U

Nucleic Acid Stain 37 X (SYBR Green I)

Cryoprotectants/Lyoprotectants 0.2-0.4 M Dextrane/ Gelatine/

Trehalose/Saccharose/Glucose

The references cited throughout the specification and examples are herein incorporated in their entirety:

1. Chakravarti, A., Rajni Kumaria, P. Kar, Vineeta Vijay Batra and Vikas Verma. 2006.

Improved Detection of Dengue Virus Serotypes from Serum Samples. Dengue Bulletin. Volume 30, p. 133-140.

2. Domingo C, Palacios G., Jabado O., Reyes N., Niedrig M., Gascon J., Cabrerizo M., Lipkin W.I., Tenorio A. 2006. Use of a short fragment of the C-terminal E gene for detection and characterization of two new lineages of dengue virus 1 in India. J Clin Microbiol. 44(4): 1519- 29. PubMed PMID: 16597885; PubMed Central PMCID: PMC1448656.

3. Yong, Y., Thayan, R., Chong, H.T., Tan, C.T. and Sekaran, S.D. 2007. Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR. Singapore Med J. 48 (7): 662.

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Claims

1. A method for the detection of a pathogen in a sample by LAMP, comprising:

Adding said sample to a reaction tube comprising of a thermostable reaction mixture and primers;

Incubating the reaction tube in a heater; and Detecting the presence of the pathogen in the sample.

2. The method in Claim 1 , wherein the thermostable reaction mixture consists of FIP, BIP, F2, B2, F3, B3, deoxynucleoside triphosphates, betaine, Tween, MgSO, KC1, TrisHCl and Bst DNA polymerase.

3. The method in Claims 1 or 2, wherein the thermostable reaction mixture is lyophilized.

4. The method in Claim 3, wherein the thermostable reaction is further mixed with water.

5. The method in Claim 1 , wherein detection is by means of adding a detection dye to the reaction tube after incubation and observing the corresponding color change in the said reaction tube.

6. The method of Claim 1 , wherein the pathogen is dengue virus.

7. The method of Claim 5, wherein the primers consist of universal LAMP primers

containing nucleic acid sequences of SEQ ID NOS. 25 -30.

8. The method of Claim 5, wherein the primers consist of dengue serotype-specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 1 -6 for serotype 1.

9. The method of Claim 5, wherein the primers consist of dengue serotype-specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 7-12 for serotype 2.

10. The method of Claim 5, wherein the primers consist of dengue serotype-specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 13-18 for serotype 3.

11. The method of Claim 5, wherein the primers consist of dengue serot pe-specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 19-24 for serotype 4.

12. The method of Claim 1 , wherein the pathogen is a Leptospira species.

13. The method of Claim 15, wherein the primers consist of LAMP primers which are

complementary to the ompLl region of the Leptospira species containing nucleic acid sequences of SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 36.

14. The method in Claim 1, wherein the pathogen is MTB or MOTT.

15. The method of Claim 17, wherein the primers consist of LAMP primers specific for the detection of MTB containing nucleic acid sequences of SEQ ID NOS. 37 - 96.

16. The method of Claim 17, wherein the primers consist of LAMP primers specific for the detection of MOTT containing nucleic acid sequences of SEQ ID NOS. 97 - 156.

17. The method of Claim 1, wherein the pathogen is Salmonella enterica.

18. The method of Claim 20, wherein the primers consist of universal LAMP primers

containing contain nucleic acid sequences of SEQ ID NOS. 157 - 192.

19. The method in Claim 20, wherein the primers consist of LAMP primers which are typhoidal serovar-specific primers containing nucleic acid sequences of SEQ ID NOS. 193 - 222.

20. A kit for the detection of a pathogen in a sample by LAMP, comprising of primers and a thermostable reaction mixture.

21. A kit as claimed in Claim 23, wherein the thermostable reaction mixture may be

lyophilized.

22. A kit as claimed in Claim 23, wherein the thermostable reaction mixture may be kept at room temperature when not in use.

23. A kit as claimed in Claim 23, wherein the thermostable reaction mixture consists of a pre- mixed mixture of FIP, BD?, F2, B2, F3, B3, deoxynucleoside triphosphates, betaine, Tween, MgSO, KC1, TrisHCl and Bst DNA polymerase.

A kit as claimed in Claim 23, wherein the kit includes a heater where the primers and the thermostable reaction mixture will be incubated at 63-67° C to achieve a LAMP isothermal temperature.

A thermostable reaction mixture for use in LAMP that would detect pathogens upon addition of a sample and primers.

A thermostable reaction mixture as claimed in Claim 23, wherein water is further added to detect pathogens.

A set of primers to detect dengue by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 25 -30.

A set of primers to detect specific serotype of dengue by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 1 -6 for serotype 1 , SEQ ID NOS. 7-12 for serotype 2, SEQ ID NOS. 13-18 for serotype 3 and SEQ ID NOS. 1 -24 for serotype 4.

A set of primers to detect Leptospira species by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NO. 31 , SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ED NO. 35 and SEQ ID NO. 36.

A set of primers to detect MTB by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 37 - 96.

A set of primers to detect MOTT by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 97 - 156.

A set of primers to detect Salmonella enterica by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 157 - 192.

A set of primers to detect typhoidal serovar Salmonella enterica, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 193 - 222.