# US 8,465,951 B2 · Method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system

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- Number: US 8,465,951 B2
- Document ID: US8465951B2
- Status: Granted
- Kind: B2
- Application number: 12/602,564
- Filed: 2008-05-27
- Published: 2011-05-12
- Granted: 2013-06-18
- Earliest claimed: 2007-06-01
- Assignees: Council of Scientific and Industrial Research
- Applicants: Chintalagiri Mohan Rao; Ikunchala Sridhar Rao; Puppala Venkat Ramchander; Hajib Naraharirao Madhavan; Savitri Sharma; Gita Satpathy; Venkata Banda Ravi Kumar
- Inventors: Chintalagiri Mohan Rao; Ikunchala Sridhar Rao; Puppala Venkat Ramchander; Hajib Naraharirao Madhavan; Savitri Sharma; Gita Satpathy; Venkata Banda Ravi Kumar
- Web page: https://patentgrep.com/patent/US8465951B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- C12Q 1/689: for bacteria
- C12Q 1/6895: for plants, fungi or algae
- C12Q 1/705: {for herpetoviridae, e.g. herpes simplex, varicella zoster}
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to the diagnostic methods for identification of the single causative agent or more than one causative agent of ocular and nervous system infections among many probable pathogens, which can cause the infection. All the pathogens affecting a discrete area of eye or nervous system generally cause same clinical manifestations or syndromes. The present invention relates to detection and discrimination of the pathogen among the set of probable pathogens in a single test without resorting to a battery of tests each being directed at detection of one pathogen. The current invention aims at the syndrome based diagnostic replacing the diagnostics based on detection of individual pathogens.

## Claims

### Claim 1

A method for the detection of pathogens in a sample comprising the steps of: amplifying specific genes of the pathogens by performing multiplex PCR (polymerase chain reaction) assay using the primer set 1 ((SEQ ID NO:1) and (SEQ ID NO:2)), set 2 ((SEQ ID NO:3) and (SEQ ID NO:4)), set 3 ((SEQ ID NO:5) and (SEQ ID NO:6)), set 4 ((SEQ ID NO:7) and (SEQ ID NO:8)), set 5 ((SEQ ID NO:9) and (SEQ ID NO:10)), set 6 ((SEQ ID NO: 11) and (SEQ ID NO:12)), set 7 ((SEQ ID NO:13) and (SEQ ID NO:14)) and set 8 ((SEQ ID NO:15) and (SEQ ID NO:16)), set 13 ((SEQ ID NO: 25) and (SEQ ID NO:26)), set 16 ((SEQ ID NO: 31) and (SEQ ID NO: 32)) and set 18 ((SEQ ID NO: 35) and (SEQ ID NO: 36)); hybridizing the amplified products (probes) to complementary DNA sequences (targets) immobilized on a solid phase matrix in a multiplex format, and detecting the hybridized products, wherein the pathogens are selected from the group consisting of Herpes simplex viruses 1 and 2, cytomegaloviruses, Varicella Zoster virus, Adenoviruses, Eubacteria, Gram positive bacteria, Gram negative bacteria, Fungi, Mycobacterium tuberculosis, Mycobacterium chelonei, Mycobacterium fortuitum, Toxoplasma gondii and Chlamydia trachomatis.

### Claim 2

A method for detection and discrimination of two or more groups of pathogens comprising the steps of
a) performing multiplex PCR (polymerase chain reaction) on DNA from a clinical sample using the combination of primer sets 1 to 22 (SEQ ID NO:1 to SEQ ID NO: 44) to obtain amplified PCR products,
b) denaturing the amplified PCR products,
c) hybridizing the denatured amplified PCR products with target DNA sequences, and
d) identifying the pathogens by detecting for hybridization.

### Claim 3 (depends on 2)

The method as claimed in claim 2, wherein the pathogen is known to cause a syndrome selected from the group consisting of infectious endophthalmitis, keratitis, uveitis, retinitis, meningitis, ocular infection, and meningoencephalitis.

### Claim 4 (depends on 2)

The method as claimed in claim 2, wherein the target DNA sequences are selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88.

### Claim 5 (depends on 3)

The method as claimed in claim 3, wherein primer sets 1 to 22 (SEQ ID NO.:1 to SEQ ID No: 44) are used together in a reaction vessel at uniform thermal-cycling conditions comprising a denaturing step of 94 C for 5 minutes, followed by 40 cycles of 45 seconds at 60° C.-64° C., 45 seconds at 72° C. for 45 seconds at 94° C. followed by 10 minutes extension of the reaction at 72° C.

### Claim 6 (depends on 2)

The method as claimed in claim 2, wherein said primers are labeled at 5′ end using a biotin moiety resulting in detection by formation of coloured product; or wherein the primers are labeled by fluorescent labels selected from organic fluorescent labels selected from a group consisting of Fluorescene isothiocyanate FITC, and inorganic fluorescent nano-particles, Quantum Dots™, Cy3, and Cy5 enabling detection by any fluorescent scanning device or microscopy.

### Claim 7 (depends on 2)

The method as claimed in claim 2, wherein the hybridization is detected using amplified PCR products comprising sequences selected from the group consisting of SED ID NO:45 to SEQ ID NO:66 as probes in a macroarray, slot blot or line probe assay.

### Claim 8 (depends on 7)

The method as claimed in claim 7, wherein the macroarray comprises target DNA sequences SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88 fixed to a solid phase selected from nitrocellulose, nylon, charged nylon, glass and polystyrene or a combination thereof.

### Claim 9 (depends on 2)

The method according to claim 2, wherein the sample is selected from the group consisting of aqueous humor, vitreous fluid, vitreous lavage, corneal scrapings, conjunctival swabs, Cerebro-spinal fluid, blood, and plasma.