Development of a real-time PCR assay for detection of pneumococcal DNA and diagnosis of pneumococcal disease

A method for detecting Streptococcus pneumoniae nucleic acid in a biological sample, comprising:

• producing an amplification product by amplifying an Streptococcus pneumoniae nucleotide sequence using sense primers and antisense primers, wherein said primers are chosen from oligonucleotides that hybridize, under conditions suitable for a polymerase chain reaction, with a sequence of the psaA gene of Streptococcus pneumoniae;
• and detecting said amplification product, whereby the presence of Streptococcus pneumoniae nucleic acid is detected,
• wherein the sense primer consists of SEQ ID NO:
• 1 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 5;
• or a sequence complementary thereto.

A method for detecting Streptococcus pneumoniae nucleic acid in a biological sample, comprising:

• producing an amplification product by amplifying an Streptococcus pneumoniae nucleotide sequence by real-time PCR using sense primers and antisense primers, wherein said primers are chosen from oligonucleotides that hybridize, under conditions suitable for a polymerase chain reaction, with a sequence of the psaA gene of Streptococcus pneumoniae;
• and
• detecting said amplification product,
• whereby the presence of Streptococcus pneumoniae nucleic acid is detected,
• wherein the antisense primer consists of SEQ ID NO:
• 2 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 6;
• or a sequence complementary thereto.

A method for detecting Streptococcus pneumoniae nucleic acid in a biological sample using Real-Time PCR, comprising:

• producing an amplification product by amplifying an Streptococcus pneumoniae nucleotide sequence using sense primers and antisense primers, wherein said primers are chosen from oligonucleotides that hybridize, under conditions suitable for a polymerase chain reaction, with a sequence of the psaA gene of Streptococcus pneumoniae;
• and
• detecting said amplification product,
• whereby the presence of Streptococcus pneumoniae nucleic acid is detected,
• wherein the nondegenerate probe consists of SEQ ID NO:
• 3 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 7;
• or a sequence complementary thereto.

An oligonucleotide comprising SEQ ID NO:

• 1 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 5;
• or a sequence complementary thereto, wherein the oligonucleotide is from 15-30 consecutive nucleotides.

An oligonucleotide, comprising at least 15 consecutive nucleotides of SEQ ID NO:

• 2 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 6;
• or a sequence complementary thereto, wherein the oligonucleotide is from 15-30 consecutive nucleotides.

An oligonucleotide comprising at least 20 consecutive nucleotides of SEQ ID NO:

• 3 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 7;
• or a sequence complementary complementary thereto, wherein the oligonucleotide is from 20-30 consecutive nucleotides.

A kit comprising reagents for real-time PCR-type amplification reaction for detecting Streptococcus pneumoniae, comprising sense primers, antisense primers and a nondegenerate probe, wherein the sense primer consists of SEQ ID NO:

• 1 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 5;
• or a sequence complementary thereto, and
• a primer consisting of SEQ ID NO:
• 2 or a sequence that hybridizes, under conditions suitable for a polymerase chain reaction, with:
• SEQ ID NO:
• 6;
• or a sequence complementary thereto,
• under conditions suitable for a polymerase chain reaction;
• and
• detecting said amplification product by using:
• a probe consisting of:
• 5′-X-CTAGCACATGC“T”ACAAGAATGATTGCAGAAAGAAA-Y-3′,
• wherein X is a fluorophore,
• wherein Y is a phosphate group or phosphate groups,
• wherein “T” is a thymine with a dark quencher or acceptor dye linked to it,
• whereby the presence of Streptococcus pneumoniae nucleic acid is detected.

A method for quantifying Streptococcus pneumoniae nucleic acid in a biological sample, comprising:

• producing an amplification product by amplifying an Streptococcus pneumoniae nucleotide sequence by real-time PCR using sense primers and antisense primers, wherein said primers are chosen from oligonucleotides that hybridize, under conditions suitable for a polymerase chain reaction, with a sequence of the psaA gene of Streptococcus pneumoniae;
• and
• detecting said amplification product by using a nondegenerate probe comprising an oligonucleotide that hybridizes, under conditions suitable for a polymerase chain reaction, with a sequence of the psaA gene of Streptococcus pneumoniae;
• and
• quantifying said amplification product in said biological sample by measuring a detection signal from said probe and comparing said detection signal to a second probe detection signal from a quantification standard, wherein said quantification standard comprises a sense probe and a nucleic acid standard.