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Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China.

Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yang Z, Huang S, Zhou D, Huang L, Yuan J - Front Microbiol (2015)

Bottom Line: Klebsiella pneumoniae is a wide-spread nosocomial pathogen.The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR.In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.

ABSTRACT
Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

No MeSH data available.


Related in: MedlinePlus

LAMP results forK. pneumoniaeisolates from clinical samples. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. 1, Negative control (double-distilled water); 2, positive control (K. pneumoniae ATCC BAA-2146); 3, K. pneumoniae WJ-48; 4, K. pneumoniae WJ-50; 5, K. pneumoniae WJ-51; 6, K. pneumoniae WJ-52; 7, K. pneumoniae WJ-53; 8, K. pneumoniae WJ-57; 9, K. pneumoniae WJ-58; 10, K. pneumoniae WJ-60; 11, K. pneumoniae WJ-61; 12, K. pneumoniae WJ-64; 13, K. pneumoniae WJ-65; 14, K. pneumoniae WJ-66; 15, K. pneumoniae WJ-68; 16, K. pneumoniae 301-052; 17, K. pneumoniae 301-207; 18, K. pneumoniae 301-263; 19, K. pneumoniae 301-432; 20, K. pneumoniae 301-416; 21, K. pneumoniae 301-323; 22, K. pneumoniae 301-365; 23, K. pneumoniae 301-282; 24, K. pneumoniae 301-406; 25, K. pneumoniae 301-158; 26, K. pneumoniae 307-206; 27, K. pneumoniae 307-082; 28, K. pneumoniae 307-429; 29, K. pneumoniae 307-095; 30, K. pneumoniae 307-003; 31, K. pneumoniae 307-030; 32, K. pneumoniae 307-194; 33, K. pneumoniae 307-356; 34, K. pneumoniae 307-235.
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Figure 5: LAMP results forK. pneumoniaeisolates from clinical samples. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. 1, Negative control (double-distilled water); 2, positive control (K. pneumoniae ATCC BAA-2146); 3, K. pneumoniae WJ-48; 4, K. pneumoniae WJ-50; 5, K. pneumoniae WJ-51; 6, K. pneumoniae WJ-52; 7, K. pneumoniae WJ-53; 8, K. pneumoniae WJ-57; 9, K. pneumoniae WJ-58; 10, K. pneumoniae WJ-60; 11, K. pneumoniae WJ-61; 12, K. pneumoniae WJ-64; 13, K. pneumoniae WJ-65; 14, K. pneumoniae WJ-66; 15, K. pneumoniae WJ-68; 16, K. pneumoniae 301-052; 17, K. pneumoniae 301-207; 18, K. pneumoniae 301-263; 19, K. pneumoniae 301-432; 20, K. pneumoniae 301-416; 21, K. pneumoniae 301-323; 22, K. pneumoniae 301-365; 23, K. pneumoniae 301-282; 24, K. pneumoniae 301-406; 25, K. pneumoniae 301-158; 26, K. pneumoniae 307-206; 27, K. pneumoniae 307-082; 28, K. pneumoniae 307-429; 29, K. pneumoniae 307-095; 30, K. pneumoniae 307-003; 31, K. pneumoniae 307-030; 32, K. pneumoniae 307-194; 33, K. pneumoniae 307-356; 34, K. pneumoniae 307-235.

Mentions: One hundred and ten clinical sputum samples were collected for LAMP-based surveillance of K. pneumoniae from ICU patients with clinically suspected multi-resistant infections in three top hospitals of Beijing. Ten sputum samples from healthy people were collected as controls. Both LAMP and PCR assay were involved to analyze the clinical samples. As shown in Figure 5, of the 110 clinical samples, LAMP detected 32 positive samples while 25 were detected by PCR. Then, 32 K. pneumoniae strains were successfully cultured from these positive samples. The healthy control samples all tested negative in each of the assays. The sequence analysis of the rcsA genes from K. pneumoniae isolates confirmed conservation with the nucleotide sequences of reported gene.


Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China.

Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yang Z, Huang S, Zhou D, Huang L, Yuan J - Front Microbiol (2015)

LAMP results forK. pneumoniaeisolates from clinical samples. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. 1, Negative control (double-distilled water); 2, positive control (K. pneumoniae ATCC BAA-2146); 3, K. pneumoniae WJ-48; 4, K. pneumoniae WJ-50; 5, K. pneumoniae WJ-51; 6, K. pneumoniae WJ-52; 7, K. pneumoniae WJ-53; 8, K. pneumoniae WJ-57; 9, K. pneumoniae WJ-58; 10, K. pneumoniae WJ-60; 11, K. pneumoniae WJ-61; 12, K. pneumoniae WJ-64; 13, K. pneumoniae WJ-65; 14, K. pneumoniae WJ-66; 15, K. pneumoniae WJ-68; 16, K. pneumoniae 301-052; 17, K. pneumoniae 301-207; 18, K. pneumoniae 301-263; 19, K. pneumoniae 301-432; 20, K. pneumoniae 301-416; 21, K. pneumoniae 301-323; 22, K. pneumoniae 301-365; 23, K. pneumoniae 301-282; 24, K. pneumoniae 301-406; 25, K. pneumoniae 301-158; 26, K. pneumoniae 307-206; 27, K. pneumoniae 307-082; 28, K. pneumoniae 307-429; 29, K. pneumoniae 307-095; 30, K. pneumoniae 307-003; 31, K. pneumoniae 307-030; 32, K. pneumoniae 307-194; 33, K. pneumoniae 307-356; 34, K. pneumoniae 307-235.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440914&req=5

Figure 5: LAMP results forK. pneumoniaeisolates from clinical samples. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. 1, Negative control (double-distilled water); 2, positive control (K. pneumoniae ATCC BAA-2146); 3, K. pneumoniae WJ-48; 4, K. pneumoniae WJ-50; 5, K. pneumoniae WJ-51; 6, K. pneumoniae WJ-52; 7, K. pneumoniae WJ-53; 8, K. pneumoniae WJ-57; 9, K. pneumoniae WJ-58; 10, K. pneumoniae WJ-60; 11, K. pneumoniae WJ-61; 12, K. pneumoniae WJ-64; 13, K. pneumoniae WJ-65; 14, K. pneumoniae WJ-66; 15, K. pneumoniae WJ-68; 16, K. pneumoniae 301-052; 17, K. pneumoniae 301-207; 18, K. pneumoniae 301-263; 19, K. pneumoniae 301-432; 20, K. pneumoniae 301-416; 21, K. pneumoniae 301-323; 22, K. pneumoniae 301-365; 23, K. pneumoniae 301-282; 24, K. pneumoniae 301-406; 25, K. pneumoniae 301-158; 26, K. pneumoniae 307-206; 27, K. pneumoniae 307-082; 28, K. pneumoniae 307-429; 29, K. pneumoniae 307-095; 30, K. pneumoniae 307-003; 31, K. pneumoniae 307-030; 32, K. pneumoniae 307-194; 33, K. pneumoniae 307-356; 34, K. pneumoniae 307-235.
Mentions: One hundred and ten clinical sputum samples were collected for LAMP-based surveillance of K. pneumoniae from ICU patients with clinically suspected multi-resistant infections in three top hospitals of Beijing. Ten sputum samples from healthy people were collected as controls. Both LAMP and PCR assay were involved to analyze the clinical samples. As shown in Figure 5, of the 110 clinical samples, LAMP detected 32 positive samples while 25 were detected by PCR. Then, 32 K. pneumoniae strains were successfully cultured from these positive samples. The healthy control samples all tested negative in each of the assays. The sequence analysis of the rcsA genes from K. pneumoniae isolates confirmed conservation with the nucleotide sequences of reported gene.

Bottom Line: Klebsiella pneumoniae is a wide-spread nosocomial pathogen.The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR.In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.

ABSTRACT
Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

No MeSH data available.


Related in: MedlinePlus